Compositions and methods for genetic analysis of polycystic kidney disease

ABSTRACT

The subject invention relates to methods for detection of mutations in a PKD gene using DHPLC. The invention includes the following aspects: identification of PKD unique sites; design of PKD-specific primers; amplification of PKD-specific products; and analysis of PCR amplified products by DHPLC. The invention further relates to compositions such as identified unique sites and PKD-specific primers, and kits for performing the methods of the invention.

FIELD OF THE INVENTION

[0001] The invention relates to a genetic testing method for identifying alterations or the absence of such alterations in a gene associated with Autosomal Dominant Polycystic Kidney Disease.

BACKGROUND OF THE INVENTION

[0002] Autosomal dominant polycystic kidney disease (ADPKD) is an exceptionally common hereditary nephropathology with an incidence of about 1 in 800 live births. The disease is progressive, phenotypically characterized by bilaterally enlarged polycystic kidneys, and typically resulting in end-stage renal disease (ESRD) by the age of 65 years. The more common complications include hypertension, macrohaematuria, urinary-tract infection, cardiac-valve abnormalities, and hernia of the anterior abdominal wall. Cyst formation is also commonly observed in the liver, although the occurrence is not associated with functional impairment of the organ. Although not as frequently reported, additional extrarenal manifestations include pancreatic cysts, connective tissue abnormalities, and cerebral-artery aneurysms.

[0003] The typical age of onset is in middle life, but the range is from infancy to 80 years. The clinical presentation of ADPKD differs between and within families as partly explained by the genetically heterogeneous nature of the disorder. Mutations in two genes, PKD-1 and PKD-2, account for nearly all cases of ADPKD (e.g., for reviews, see Arnaout, 2001, Annu Rev. Med. 52:93-123; Koptides and Deltas, 2000, Hum. Genet. 107:115-126). PKD-1 and PKD-2 encode integral membrane proteins whose functions have not been fully elucidated. The major gene responsible for ADPKD, PKD-1, has been fully characterized and shown to encode an integral membrane protein, polycystin 1, which is thought to be involved in cell-cell and cell-matrix interaction. PKD-2 gene encodes polycystin-2 which is a predicted integral membrane protein with non-selective cation channel activity. Based on sequence homology with the alpha 1 subunit component of voltage-activated calcium channels, it has been postulated that polycystin-2 may play a role in ion channeling. The C-terminal cytoplasmic tails of polycystin-1 and polycystin-2 have been shown to interact using in vitro binding assays and in a directed two-hybrid interaction. The interaction occurs via a coiled-coil domain in PKD-1 and a region near R872 in PKD-2. Although the biological relevance of the interaction between the polycystins is not yet understood, it does suggest that PKD-1 and PKD-2 are likely to function along a common pathway.

[0004] Both ADPKD type 1 and type 2 share the entire range of renal and extrarenal manifestations, but type 2 appears to have a delayed onset relative to type 1. The common phenotypic complications observed for ADPKD including hypertension, hematuria, and urinary tract infection seem to be clinically milder in type 2 patients. The median age at death or onset of ESRD has been reported as 53 years in individuals with PKD-1 and 69 years in those with PKD-2. Women have been reported to have a significantly longer median survival of 71 years than men (67 years). No sex influence is apparent in PKD-1. Mutations in the PKD-1 gene are the cause of ADPKD in approximately 85% of the cases tested, while those in PKD-2 account for 15%. Although a small subset of families with ADPKD fail to demonstrate genetic linkage to either PKD-1 or PKD-2, raising the possibility of a third gene for ADPKD, the existence of a third disease-associated locus has been strongly challenged.

[0005] Despite the discovery of strong links between genetic alterations in PKD genes and the onset of ADPKD, the development of a genetic testing method for ADPKD predisposition for routine clinical use has been hindered by several technical obstacles.

[0006] One serious obstacle for developing a DNA-based testing method for ADPKD is that sequences related to the PKD transcript, for example, PKD-1, are duplicated at least three times on chromosome 16 proximal to the PKD-1 locus, forming PKD-1 homologues. Another obstacle is that the PKD-1 genomic interval also contains repeat elements that are present in other genomic regions. In addition, the sequences of PKD genes are extremely GC rich and a large number (15,816 bp) of nucleotides need to be analyzed for a thorough evaluation.

[0007] There is a need for the identification of segments of these sequences that are unique to the expressed PKD genes and not are present in the duplicated homologous sequences. There is also a need for developing a sensitive and specific genetic testing method for mutational analysis of PKD genes. The development of such genetic testing method would facilitate the diagnosis and management of ADPKD.

SUMMARY OF THE INVENTION

[0008] In one aspect, the present invention provides a method of mutation analysis of a target nucleic acid, the method comprising:

[0009] incubating a sample comprising the target nucleic acid in a reaction mixture, in the presence of at least one first nucleic acid and at least one second nucleic acid, where the first nucleic acid comprises a primer sequence which anneals to a unique site of a sequence of SEQ ID NO. 1 or 2, and the second nucleic acid has an opposite orientation from the first nucleic acid, and where the incubation produces amplified products;

[0010] generating duplexes in the amplified products; and

[0011] detecting the presence or absence of a heteroduplex from the duplexes, where the presence of a heteroduplex indicates the presence of a potential mutation in the target nucleic acid, and where the absence of a heteroduplex indicates the absence of a mutation in the target nucleic acid.

[0012] In one embodiment, the method further comprises determining the sequence of a heteroduplex region; and comparing the sequence of the heteroduplex region to SEQ ID NO. 1 or 2; where a sequence difference in the heteroduplex region compared to SEQ ID NO. 1 or 2 resulting in a predicted functional change in the protein encoded by the target nucleic acid is indicative of a mutation in the target nucleic acid.

[0013] Preferably, the first or second nucleic acid comprises a sequence selected from the group consisting of SEQ ID NOs. 3-49.

[0014] In another embodiment, the method further comprising performing a nested amplification reaction using the amplified products generated by the first and second nucleic acids as templates and generating duplexes in amplified products from the nested amplification.

[0015] Preferably, the nested amplification reaction is performed using at least one primer selected from the group consisting of SEQ ID NOs. 3-49 and their complementary sequences.

[0016] In a preferred embodiment, the presence or absence of a heteroduplex from the duplexes is identified by DHPLC.

[0017] In also a preferred embodiment, the sequence of the heteroduplex region is determined by DNA sequencing.

[0018] Preferably, the second nucleic acid of the subject method comprises a primer sequence which anneals to a unique site within a sequence of SEQ ID NO. 1 or 2.

[0019] Also preferably, the sample comprising the target template is selected from the group consisting of: genomic DNA, cDNA, total RNA, mRNA, and a cell sample.

[0020] In one embodiment, the incubating step comprises an amplification reaction selected from the group consisting of: a polymerase chain reaction, a ligase chain reaction (LCR) and a nucleic acid-specific based amplification.

[0021] The subject method of the invention may further comprise confirming the amplified product is a PKD-specific product with one or more restriction enzymes.

[0022] Preferably, the restriction enzyme cleaves a PKD-specific product to generate a digestion pattern distinguishable from a PKD homologue product.

[0023] More preferably, the restriction enzyme is selected from the group consisting of: Pst I, Stu I, Xma I, Mlu I, Pvu II, BssHII, Fsp I, Msc I, and Bln I.

[0024] In another aspect, the invention provides a diagnosis method for identifying a patient affected with PKD, the method comprising:

[0025] (a) obtaining a sample from an individual;

[0026] (b) incubating the sample in a reaction mixture, in the presence of at least one first nucleic acid and at least one second nucleic acid, where the first nucleic acid comprises a primer sequence which anneals to a unique site within a sequence of SEQ ID NO. 1 or 2, and the second nucleic acid has an opposite orientation from the first nucleic acid, and where the incubation produces amplified products;

[0027] (c) generating duplexes in the amplified products;

[0028] (d) detecting the presence or absence of a heteroduplex from the duplexes, and(e) determining the sequence of the heteroduplex region where the presence of a mutation in the heteroduplex region as compared to SEQ ID No. 1 or 2 is indicative that the individual is affected with PKD.

[0029] Preferably, the detection of a heteroduplex is performed by DHPLC.

[0030] Also preferably, the sequence is determined by DNA sequencing.

[0031] In one embodiment, the second nucleic acid comprises a primer sequence which anneals to a unique site within a sequence of SEQ ID NO. 1 or 2.

[0032] In another embodiment, the first or second nucleic acid comprises a primer sequence selected from the group consisting of SEQ ID NOs. 3-49.

[0033] The diagnosis method of the invention may further comprise performing a nested amplification reaction using the amplified products generated by the first and second nucleic acids as templates and generating duplexes from the nested amplification.

[0034] In one embodiment, the nested amplification reaction is performed using at least one primer selected from the group consisting of SEQ ID NOs. 3-49 and their complementary sequences.

[0035] Preferably, the sample in the diagnosis method is selected from the group consisting of: a genomic DNA, cDNA, total RNA, mRNA, and a cell.

[0036] Also preferably, the amplification reaction is selected from the group consisting of: a polymerase chain reaction, a ligase chain reaction (LCR) and a nucleic acid-specific based amplification.

[0037] The diagnosis method may further comprise verifying the specifically amplified product with one or more restriction enzymes.

[0038] Preferably, the restriction enzyme cleaves a PKD-specific product to generate a digestion pattern distinguishable from a PKD homologue product.

[0039] More preferably, the restriction enzyme is selected from the group consisting of: Pst I, Stu I, Xma I, Mlu I, Pvu II, BssHII, Fsp I, Msc I, and Bln I.

[0040] In a further aspect, the invention provides one or more nucleic acid primer, where each primer is an isolated nucleic acid selected from the group of SEQ ID NOs 3-49, or the complement thereof.

[0041] The invention also provides a pair of nucleic acids, where at least one nucleic acid of the pair is selected from the group of SEQ ID NOs 3-49.

[0042] Preferably, the pair of nucleic acids have an opposite orientation and amplify a fragment of a template nucleic acid comprising a sequence of SEQ ID NO. 1 or 2.

[0043] In another aspect, the invention provides a composition comprising at least one isolated first nucleic acid and at least one isolated second nucleic acid, where the first nucleic acid is selected from the group of SEQ ID NOs. 3-49 and their complementary sequences, and the second nucleic acid has an opposite orientation from the first nucleic acid, and wherein the first and second nucleic acids amplify a fragment of a template nucleic acid comprising a sequence of SEQ ID NO. 1 or 2.

[0044] In one embodiment, the composition of the invention further comprises at least one component selected from the group consisting of: a DNA polymerase, a template nucleic acid, a restriction enzyme, one or more control oligonucleotide primers, ddNTPs, a PCR reaction buffer and their combination thereof.

[0045] Preferably, the template nucleic acid in the composition is a genomic DNA or cDNA.

[0046] In a further aspect, the invention provides a kit for identifying a PKD patient, the kit comprising at least one isolated first nucleic acid and at least one isolated second nucleic acid, where the first nucleic acid is selected from the group of SEQ ID NOs. 1-49 and their complementary sequences, and the second nucleic acid has an opposite orientation from the first nucleic acid, and where the first and second nucleic acids amplify a fragment of a template nucleic acid comprising a sequence of SEQ ID NO. 1 or 2, and packaging materials therefore.

[0047] In one embodiment, the kit of the invention further comprises at least one component selected from the group consisting of: a DNA polymerase, a template nucleic acid, a restriction enzyme, a control oligonucleotide primer, ddNTPs, a PCR reaction buffer and the combination thereof.

[0048] Preferably, the template nucleic acid in the kit is a genomic DNA or cDNA molecule.

BRIEF DESCRIPTION OF THE DRAWINGS

[0049] The objects and features of the invention can be better understood with reference to the following detailed description and accompanying drawings.

[0050]FIG. 1 is a figure showing the PKD1 cDNA sequence (GenBank Accesion No. L33243) used in one embodiment of the invention. Exon and PCR product junctions are depicted above the nucleotide sequence. Amino acids are positioned under the center of each codon.

[0051]FIG. 2 is a figure showing the comparison of exon sequences of a PKD gene and two homologue sequences according to one embodiment. Restriction enzyme sites which only cleave in either PKD or homologue sequence are indicated.

[0052]FIG. 3 is a graph showing PKD1 exon 40 DHPLC patterns of 4 normal samples and a 19 bp insertion (duplication) at nucleotide 11606, codon 3799 according to one embodiment.

[0053]FIG. 4 is a graph showing PKD1 exon 40 sequences of the normal control and a sequence with a 19 bp insertion (duplication) at nucleotide 11606, codon 3799 according to one embodiment.

[0054]FIG. 5 is a graph showing PKD1 exon 6 DHPLC patterns of an intron 5 probable polymorphism (IVS5-9 G->A) and a frameshift at nucleotide 1502 (insert G) in two related patients according to one embodiment.

[0055]FIG. 6 is a graph showing PKD1 exon 6 sequences of the normal control and a sequence with intron 5 probable polymorphism (IVS5-9 G->A) according to one embodiment.

[0056]FIG. 7 is a graph showing PKD 1 exon 18 DHPLC patterns of a frameshift at nucleotide 7518, codon 2436 (insert C), and a common polymorphism C7652T according to one embodiment.

[0057]FIG. 8 is a graph showing PKD1 exon 18 sequences of the normal control and a sequence with frameshift at nucleotide 7518, codon 2436 (insert C) according to one embodiment.

[0058]FIG. 9 is a graph showing an example of a software-predicted melt profile and the need for multiple temperatures to establish partial melting near the ends of an exon according to one embodiment.

[0059]FIG. 10A is a chart showing patient DNA variant genotypes determined in one embodiment of the invention.

[0060]FIG. 10B is a table showing patient DNA variant genotypes determined in one embodiment of the invention.

[0061]FIG. 11 is a table summarizing DHPLC (WAVE) conditions used in some embodiments of the invention.

[0062]FIG. 12 is a table summarizing PCR conditions used in some embodiments of the invention.

[0063]FIG. 13 is a schematic diagram showing patient specimen processing steps in one embodiment of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0064] The subject invention is based on the identification of unique sites within a PKD gene, the design of PKD-specific primers and the DHPLC analysis of PCR products amplified by using these PKD-specific primers.

[0065] I. Definitions

[0066] As used herein, “ADPKD” refers to autosomal dominant polycystic kidney disease. ADPKD is an exceptionally common hereditary nephropathology and is characterized by the development of renal cysts and, ultimately, renal failure, and may alternatively or in addition involve cysts in other organs including liver and spleen, as well as gastrointestinal, cardiovascular, and musculoskeletal abnormalities.

[0067] The term “PKD gene” refers to a genomic DNA sequence which maps to chromosomal position 16p13.3 (i.e., PKD-1) or chromosomal position 4q21-23 (i.e., PKD-2) and gives rise to a messenger RNA molecule encoding a PKD protein. The PKD-1 and PKD-2 genes comprise the sequences of SEQ ID NO. 1 and SEQ ID NO.2, respectively, which include introns and putative regulatory sequences. Like many other genes, PKD-1 and PKD-2 gene sequences, when compared among individuals, show sequence variations. Those genes having polymorphisms which are silent (i.e., with respect to gene expression or function of a gene product) are “normal” genes as defined herein.

[0068] A “normal” PKD gene (e.g., PKD-1 or PKD-2) is defined herein as a PKD gene such as described by SEQ ID NO. 1 or 2, respectively, and includes any gene having silent polymorphisms.

[0069] A “mutant” PKD gene is defined herein as a PKD gene (e.g., PKD-1 or PKD-2) whose sequence is modified by mutation comprising one or more substitutions (transitions or transversions), deletions (including loss of locus), insertions (including duplications), translocations, and/or other modifications relative to the normal PKD gene. The mutation causes detectable changes in the expression or function of the PKD gene product, and is causative for ADPKD. The mutations may involve from one to as many as several thousand nucleotides, and result in one or more of a variety of changes in PKD gene expression (e.g. decreased or increased rates of expression) or expression of a defective RNA transcript or protein product. Mutant PKD genes encompass those genes whose presence in one or more copies in the genome of a human individual is associated with ADPKD.

[0070] The term “basepair mismatch” refers to any nucleic acid sequence which is not complementary to the sequence of SEQ ID. NO. 1 or 2. Therefore, basepair mismatch, according to the present invention may be caused by gene alteration or polymorphism of a normal PKD gene; or by any modifications present in a mutant PKD gene. “Basepair mismatch” may be a single nucleotide basepair mismatch or it may include a nucleic acid sequence of 2 or more nucleotides (i.e., 3, or 4, or 5, or 10, or 20, or 100, or 500 more, or up to 1000 nucleot The presence or absence of a mismatch, as defined herein, is indicative of the presence or absence of a potential mutation in the target nucleic acid.

[0071] The term “authentic” is used herein to denote the genomic sequence of SEQ ID. NO. 1 or 2, as well as sequences derived therefrom, and serves to distinguish these authentic sequences from “PKD homologues” (see below).

[0072] A “PKD-1 homologue” is a sequence which is closely related to PKD-1, but which does not encode an expressed PKD-1 gene product. Several examples of such homologues that map to chromosomal location 16p13.1 or 4q21-23 have been identified and sequenced. A PKD-1 homologue may share more than 95% sequence identity to an authentic PKD gene.

[0073] As used herein, a “specifically amplified product” is a product amplified from a fragment within an authentic PKD gene (e.g., SEQ ID NO. 1 or 2), but not from a PKD homologue. A “non-specifically amplified product” is a product amplified from a PKD homologue or other sequences due to the annealing of nucleic acid primers to a template sequence which is not completely complementary during the amplification reaction.

[0074] As used herein, a “unique site” refers to a stretch of sequence of 10-50 base pairs in length within a PKD gene which comprises at least one nucleotide different form a stretch of sequence in a PKD homologue or other sequences. One exemplary unique site comprises a sequence of 5′ AGG TCC AGG GCG ACT CGC TGG 3′, or 5′ CAG GGC CAC ACG CGC TGG GCG 3′, or their complement thereof.

[0075] As used herein, a “PKD-specific primer” refers to a nucleic acid sequence which anneals to a sequence within a PKD gene (including introns and exons) under specific stringent conditions. A PKD-specific primer, according to the invention, anneals to a unique site present in the authentic expressed PKD-1 gene or PKD-2 gene, and not to PKD homologues or other sequences under specific stringent conditions. A PKD-specific primer shares more then 95% (e.g., more than 96%, 96%, 97%, 98%, 99%, or up to 100%) sequence identity with a unique site within a PKD gene. A “PKD-specific primer” may be 10 to 60 nucleotides in length, for example, 18-52 nucleotides in length.

[0076] As used herein, the term “specific stringent condition” refers to an amplification condition which specifically allows the annealing of a PKD-specific primer to a sequence within a PKD gene. Under a “specific stringent condition”, a PKD-specific primer does not anneal to a PKD homologue or other sequences. For example, one specific stringent condition useful to the invention comprises a Taq Precision buffer (TaqPlus Precision buffer, Stratagene, La Jolla, Cat# 600210), a dNTP concentration of more than 50 nM, for example, 100 nM, 200 nM, or 300 nM. The annealing temperature in a specific stringent condition may be higher than or less than or equal to 5° C. below the lowest primer annealing temperature (Tm), for example, 1° C., 2° C., 4° C., 5° C., or 10° C. higher than Tm or 4° C., 3° C., 2° C., or 1° C. below Tm.

[0077] “Amplification” of DNA as used herein refers to a reaction that serves to increase the concentration of a particular DNA sequence within a mixture of DNA sequences. Amplification may be carried out using polymerase chain reaction (PCR), ligase chain reaction (LCR), nucleic acid-specific based amplification (NSBA), or any other method known in the art.

[0078] “RT-PCR” as used herein refers to coupled reverse transcription and polymerase chain reaction. This method of amplification uses an initial step in which a specific oligonucleotide, oligo dT, or a mixture of random primers is used to prime reverse transcription of RNA into single-stranded cDNA; this cDNA is then amplified using standard amplification techniques e.g. PCR.

[0079] A “template nucleic acid” or a “target nucleic acid” (e.g., a genomic DNA or a cDNA), is a normal (e.g., wild type) or a mutant nucleic acid that is or includes a particular sequence (e.g. a PKD-1 or PKD-2 gene sequence). It will be understood that additional nucleotides may be added to the 5′ and/or 3′ terminus of the disclosed sequence, as part of routine recombinant DNA manipulations. Furthermore, conservative DNA substitutions i.e. changes in the sequence of the protein-coding region that do not change the encoded amino acid sequence, also may be accommodated.

[0080] As used herein, “nucleic acid primer” refers to a DNA or RNA molecule capable of annealing to a nucleic acid template and providing a 3′ end to produce an extension product which is complementary to the nucleic acid template. The nucleic acid template is catalyzed to produce a primer extension product which is complementary to the target nucleic acid template. The conditions for initiation and extension include the presence of four different deoxyribonucleoside triphosphates and a polymerization-inducing agent such as DNA polymerase or reverse transcriptase, in a suitable buffer (“buffer” includes substituents which are cofactors, or which affect pH, ionic strength, etc.) and at a suitable temperature. The primer according to the invention may be single or double stranded. The primer is single-stranded for maximum efficiency in amplification, and the primer and its complement form a double-stranded nucleic acid. But it may be double stranded. “Primers” useful in the present invention are less than or equal to 100 nucleotides in length, e.g., less than or equal to 90, or 80, or 70, or 60, or 50, or 40, or 30, or 20, or 15, or equal to 10 nucleotides in length.

[0081] As used herein, the term “opposite orientation”, when referring to primers, means that one primer comprises a nucleotide sequence complementary to the sense strand of a target nucleic acid template, and another primer comprises a nucleotide sequence complementary to the antisense strand of the same target nucleic acid template. Primers with an opposite orientation may generate a PCR amplified product from matched nucleic acid template to which they complement. Two primers with opposite orientation may be referred to as a reverse primer and a forward primer.

[0082] As used herein, the term “same orientation”, means that primers comprise nucleotide sequences complementary to the same strand of a target nucleic acid template. Primers with same orientation will not generate a PCR amplified product from matched nucleic acid template to which they complement.

[0083] Alternatively, primers of the present invention may be labeled with a detectable label such as a radioactive moiety, or a fluorescent label, or alternatively, the amplification reaction may incorporate labeled nucleotides into the reaction product. Thus, the amplification reaction product may be “detected” by “detecting” the fluorescent or radioactive label.

[0084] As used herein, a “nucleic acid” generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. “Nucleic acids” include, without limitation, single- and double-stranded nucleic acids. As used herein, the term “nucleic acid(s)” also includes DNAs or RNAs as described above, that contain one or more modified bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are “nucleic acids”. The term “nucleic acids” as it is used herein embraces such chemically, enzymatically or metabolically modified forms of nucleic acids, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including for example, simple and complex cells.

[0085] As used herein, “isolated” or “purified” when used in reference to a nucleic acid means that a naturally occurring sequence has been removed from its normal cellular (e.g., chromosomal) environment or is synthesized in a non-natural environment (e.g., artificially synthesized). Thus, an “isolated” or “purified” sequence may be in a cell-free solution or placed in a different cellular environment. The term “purified” does not imply that the sequence is the only nucleotide present, but that it is essentially free (about 90-95%, up to 99-100% pure) of non-nucleotide or nucleic acid material naturally associated with it, and thus is distinguished from isolated chromosomes.

[0086] As used herein, “genomic DNA” refers to chromosomal DNA, as opposed to complementary DNA copied from an RNA transcript. “Genomic DNA”, as used herein, may be all of the DNA present in a single cell, or may be a portion of the DNA in a single cell.

[0087] As used herein, “complementary” refers to the ability of a single strand of a nucleic acid (or portion thereof) to hybridize to an anti-parallel nucleic acid strand (or portion thereof) by contiguous base-pairing between the nucleotides (that is not interrupted by any unpaired nucleotides) of the anti-parallel nucleic acid single strands, thereby forming a double-stranded nucleic acid between the complementary strands. A first nucleic acid is said to be “completely complementary” to a second nucleic acid strand if each and every nucleotide of the first nucleic acid forms base-pairing with nucleotides within the complementary region of the second nucleic acid. A first nucleic acid is not completely complementary to the second nucleic acid if one nucleotide in the first nucleic acid does not base pair with the corresponding nucleotide in the second nucleic acid.

[0088] As used herein, a “sample” refers to a biological material which is isolated from its natural environment and containing target nucleic acid, and may consist of purified or isolated nucleic acid, or may comprise a biological sample such as a tissue sample, a biological fluid sample, or a cell sample comprising target nucleic acid.

[0089] As used herein, a “double stranded DNA” is referred to as a “duplex”. When the base sequence of one strand is entirely complementary to base sequence of the other strand, the duplex is called a “homoduplex”. When a duplex contains at least one base pair which is not complementary, the duplex is called a “heteroduplex”. In the subject invention, the formation of a heteroduplex, when amplified products from a sample taken from an individual are denatured and re-annealed, indicates the presence of a potential mutant PKD gene in that individual.

[0090] As used herein, “DHPLC” refers to a separation process called “denaturing high performance liquid chromatography” which has been used to detect sequence variants by separating a heteroduplex (resulting from the presence of a mutation) and a homoduplex having the same bp length. This separation is based on the fact that a heteroduplex has a lower melting temperature (Tm) than a homoduplex. DHPLC can separate heteroduplexes that differ by as little as one base pair under certain conditions. DHPLC can also be used to separate duplexes having different bp in length.

[0091] The “heteroduplex site separation temperature” or “midpoint temperature” or “Tm” is defined herein to mean, the temperature at which one or more base pairs denature, i.e., separate, at the site of base pair mismatch in a heteroduplex DNA fragment.

[0092] II. General Description of PKD Genes

[0093] The PKD-1 gene (e.g., genbank accession number L39891, SEQ ID NO. 1) spans about 54 kb of genomic DNA on chromosome 16 (16p13.3) and contains a 12,906 bp coding sequence divided into 46 exons from which a 14 kb mRNA is transcribed (Mochizuki et al., 1996, Science, 272:1339-1342; Hughes et al., 1995, Nature Genet. 10:151-160). The protein product of PKD-1, Polycystin-1, is a 4303 amino acid protein with a predicted mass of 460 kDa. Until recently, analysis of the PKD-1 gene had not been amenable to genetic analysis largely because of the presence of at least three highly homologous copies of the gene that map proximal to PKD-1 along chromosome 16 (16p13.1). Approximately 75% of the PKD-1 gene is duplicated and shares about 97% identity with its homologous copies. The reiterated region encompasses a 50 kb (5′) portion of the gene containing the first 33 exons. Only the most 3′, 5.7 kb of the gene, containing exons 34-46, is unique to PKD-1. Another notable feature of the PKD-1 gene is a polypyrimidine tract in intron 21 that is 2.5 kb long, the longest described in the human genome. The PKD-2 gene (e.g., genbank accession number AF004859-004873, SEQ ID NO. 2) spans 68 kb of genomic DNA and is located on chromosome 4 (4q21-23) (Mochizuki et al., 1996, supra). PKD-2 contains 15 exons and encodes a 5.4 kb transcript from which a 968-amino acid protein product of approximately 110 kDa is generated. Mutation analysis of PKD-2 is to a great extent easier than that of PKD-1 because PKD-2 is a single copy gene. See Table 1 for a summary of PKD genes and their protein products. TABLE 1 PKD gene description Gene Description PKD-1 PKD-2 Chromosome 16p13.3 4q21-23 Genomic length 54 kb 68 kb Exons 46 15 Base pairs 12909 2904 Codons 4303 968 Protein Polycystin-1 Polycystin-2

[0094] Based on evidence supporting the occurrence of somatic mutations on the normal allele, a two-hit model similar to the pathogenesis of the many familial cancer predisposition syndromes has been proposed to explain the clinically focal manifestations of the disease (Qian et al., 1996, Cell, 87:979-987; Watnick et al., 1998Mol. Cell. 2:247-251). Briefly, the model suggests that ADPKD is recessive at the cellular level and that a second somatic mutation or “hit” in a heterozygous PKD defective background would result in the homozygous loss of PKD function in the affected renal tubular epithelial cell. The loss of PKD function is postulated to disrupt the signaling mechanisms required for proper cell differentiation and in turn leads to the abnormal proliferation of the afflicted cell into cystic structures.

[0095] Direct sequencing of the PKD-1 gene has revealed the presence of polymorphism in normal individuals and a multitude of different sequence alterations in ADPKD affected individuals. Table 2 shows a sypnosis of the PKD-1 sequence alterations described in the literature to date. TABLE 2 Published pkd-1 sequence alterations including mutations and polymorphisms* Codon Nucleotide Nucleotide Amino Acid Number Number Fragment number Change Change Consequence Intron 1-Exon 5 3kb del 5 224  1 13del frameshift 88 474  2 GCG-GTG Ala-Val 92 487  2 GCG-GCA Ala-Ala polymorphism 225 885  5A + 5B TCG-TAG Ser-X termination 227 890  5A + 5B CAG-TAG Gln-X termination 230 900  5A + 5B TGC-TTC Cys-Phe 324 1182  5B + 5C CGC-CTC Arg-Leu 341 1234  5C GCC-GCT Ala-Ala polymorphism 373 1330  5C CTT-CTC Leu-Leu polymorphism 403 1420  6 CAC-CAT His-His polymorphism  7 CAG-CAA splice acceptor skip exon 7 570 1921  8 CAC-CAT His-His polymorphism  9 CAG-CAT splice acceptor skip exon 9 695 2296 10 C del = ccc-cc{circumflex over ( )}g Pro-Pro frameshift 695 2296 10 C ins = ccc-cc{circumflex over ( )}c Pro-Pro frameshift 705 2324 11A CAG-TAG Gln-X termination 738 2425 11A CCC-CCG Pro-Pro polymorphism 749 2457 11A TCA-TGA Ser-X termination 845 2745 11B TTG-TCG Leu-Ser 898 2905 11B + 11C GCA-GCC Ala-Ala polymorphism 900 2911 11B + 11C CCG-CCA Pro-Pro polymorphism 910 2941 11B + 11C GAC-GAT Asp-Asp polymorphism 967 3110 12 TGG-CGG Trp-Arg 991 3183 12 GTC-GGC Val-Val polymorphism 13 AGC-TGC splice acceptor skip exon 13 1003 3220 13 4bp del = agc-ag{circumflex over ( )}g Ser-Arg frameshift 1021 3274 13 GGT-GGC Gly-Gly polymorphism 1037 3322 13 CTA-CTG Leu-Leu polymorphism 1041 3336 13 del g = ggc-g{circumflex over ( )}cg Gly-Ala frameshift 14 AGG-AAG splice acceptor skip exon 14 1092 3486 14 CAT-CAC His-His polymorphism 1124 3583 15A GCC-GCT Ala-Ala polymorphism 1125 3586 15A TCC-TCT Ser-Ser polymorphism 1166 3707 15A + 15B GGC-AGC Gly-Ser probable path. 1198 3804 15B 7bp del = agc-a{circumflex over ( )}gg Ser-Arg frameshift 1288 4075 15C + 15D CAC-CAT His-His polymorphism 1289 4077 15C + 15D t del = gtg-g{circumflex over ( )}gc Val-Gly frameshift 1309 4137 15D ct del = cct-c{circumflex over ( )}ga Pro-Arg frameshift 1346 4249 15D ac del = aca-ac{circumflex over ( )}a Thr-Thr frameshift 1360 4291 15D + 15E g del = gtg-gt{circumflex over ( )}c Val-Val frameshift 1399 4406 15E TGG-CGG Trp-Arg 1525 4784 15G g del = gtt-{circumflex over ( )}tta Val-Leu frameshift 1537 4820 15G GAG-TAG Glu-X termination 1545 4846 15G AAG-AAA Lys-Lys polymorphism 1555 4876 15G + 15H GCA-GCC Ala-Ala polymorphism 1558 4885 15G + 15H ACG-ACA Thr-Thr polymorphism 1960 6089 15L CAG-TAG Gln-X termination 1992 6187 15L 4bp del = ttc-tt{circumflex over ( )} ** frameshift 1995 6195 15L CGC-CAC Arg-His polymorphism 2039 6326 15M + 15L CAG-TAG Gln-X termination 2075 6434 15M 28bp del frameshift 2144 6642 15M 27bp del frameshift 2163 6698 15M CGA-TGA Arg-X termination 2192 6785 15M + 15N 7bp del = acc-{circumflex over ( )}gct Thr-Ala frameshift 2220 6868 15N 15bp del = cgg-{circumflex over ( )}gtg Arg-Val in frame deletion 2222 6876 15N GCG-GTG Ala-Val 2229 6898 15N TGC-TGA Cys-X termination 2242 6937 15N ac del = aca-ac{circumflex over ( )}a Thr-Thr frameshift 2243 6938 15N CAG-TAG Gln-X termination 2250 6960 15N ACG-ATG Thr-Met 15 GGT-GGG splice donor 16 CAG-GAG splice acceptor skip exon 16 2309 7138 16 GGC-GGT Gly-Gly polymorphism 2113 7147 16 GCG-GCA Ala-Ala polymorphism 2323 7179 16 14bp del = gtc-gt{circumflex over ( )} Val-X termination 2329 7196 16 CGG-TGG Arg-Trp 2332 7205 16 7del = gct-{circumflex over ( )}tgg Ala-Trp frameshift 2334 7211 16 7ins = gtg-{circumflex over ( )}gtg Val-Val frameshift 2336 7219 16 TAC-TAA Tyr-X termination 17 CAG-GAG splice acceptor skip exon 17 2370 7321 17 TGT-TGA Cys-X termination 2371 7324 17 gt del = gtg-gt{circumflex over ( )}c Val-Val frameshift 2378 7345 17 GTG-GTT Val-Val polymorphism 2379 7347 17 TAC-TGC Tyr-Cys 2389 7376 17 TTG-CTG Leu-Leu polymorphism 2392 7386 17 CGC-CCC Arg-Pro 2396 7397 17 11bp ins = att-{circumflex over ( )}ttg Ile-Leu frameshift 2402 7415 17 CGA-TGA Arg-X termination 2408 7433 18 CGT-TGT Arg-Cys probable path. 2423 7479 18 TCC-TTC Ser-Phe 2430 7499 18 CGA-TGA Arg-X termination 2442 7535 18 3bp ins = gag-g{circumflex over ( )}gcg Glu-Gly probable path. 2471 7623 18 CCG-CTG Pro-Leu 2481 7652 18 CTG-TTG Leu-Leu polymorphism 2495 7696 18 TGC-TGT Cys-Cys polymorphism 2519 7767 19 CAG-CTG Gln-Leu 2548 7853 19 GAG-CAG Glu-Gln polymorphism 2558 7883 19 CAG-TAG Gln-X termination 2570 7919 20 TTG-CTG Leu-Leu polymorphism 2579 7945 20 ggc del = ggc-{circumflex over ( )}agc Gly-Ser Gly del in frame 2582 7956 20 ACG-ATG Thr-Met polymorphism 2597 8002 20 CCA-CCG Pro-Pro polymorphism 2604 8021 20 GAT-AAT Asp-Asn polymorphism 2607 8030 20 5bp del = cac-{circumflex over ( )}cat His-His frameshift 2612 8046 20 gtt del = tcgtt-tc{circumflex over ( )}g Ser-Ser Leu del in frame 2638 8124 21 CAC-CGC His-Arg polymorphism 2639 8126 21 CGA-TGA Arg-X termination 2639 8126 21 20 ins = cga-c{circumflex over ( )}**** Arg-** frameshift 2649 8157 21 ACT-ATT Thr-Ile 2650 8159 21 del ct = ctg-{circumflex over ( )}ggt Leu-Gly frameshift 2658 8183 21 8bp del Val-X termination 2674 8231 22 CCC-TCC Pro-Ser polymorphism 2696 8298 22 CTC-CGC Leu-Arg 2708 8334 22 ACG-ATG Thr-Met polymorphism 2734 8411 23A CCA-ACA Pro-Thr polymorphism 2735 8415 23A CAG-CTG Gln-Leu polymorphism 2745 8446 23A TCT-TCG Ser-Ser polymorphism 2760 8490 23A ATG-ACG Met-Thr 2761 8493 23A CGC-CCC Arg-Pro 2763 8498 23A CTC-GTC Leu-Val 2764 8502 23A ATG-ACG Met-Thr 2765 8504 23A CGC-TGC Arg-Cys polymorphism 2766 8507 23A 12bp ins/dup in frame mutation 2782 8556 23A GTG-ATG Val-Met polymorphism 2791 8583 23A + 23B CGG-CAG Arg-Gln 2813 8650 23A + 23B AGC-AGT Ser-Ser polymorphism 2814 8651 23A + 23B GGG-AGG Gly-Arg polymorphism 2815 8657 23A + 23B c del = gcc-g{circumflex over ( )}cc Ala-Ala frameshift 2826 8688 23B + 23C ATC-ACC Ile-Thr 2888 8873 23C CGC-GGC Arg-Gly polymorphism 2893 8890 23C TCC-TCG Ser-Ser polymorphism 2900 8909 23C CAG-TAG Gln-X termination 2905 8924 23C GTC-ATC Val-Ile polymorphism 2921 8973 23C CAT-CCT His-Pro 2966 9109 24 GAG-GAC Glu-Asp polymorphism 2971 9124 24 GCT-GCC Ala-Ala polymorphism 2972 9125 24 GAC-AAC Asp-Asn polymorphism 2978 9142 24 ttc del del of Phe in frame deletion 2985 9164 25 AGA-GGA Arg-Gly 2988 9175 25 GCG-GCA Ala-Ala polymorphism 2993 9189 25 CTG-CCG Leu-Pro probable path. 3001 9213 25 TGG-TAG Trp-X termination 3008 9233 25 GTG-CTG Val-Leu 3012 9245 25 18bp del in frame deletion 3016 9258 25 CAG-CGG Gln-Arg probable path. 3020 9269 25 GAG-TAG Glu-X termination 3030 9299 25 c del = ctg-{circumflex over ( )}tgc Leu-Cys frameshift 2985 9326 25 CGC-TCG Arg-Cys 3052 9367 25 GGC-GGT Gly-Gly polymorphism 3064 9401 25 TTT-CTT Phe-Leu 3065 9406 25 GTTT-CCTT Phe-Leu polymorphism 3065 9406 25 GTG-GTC Val-Val polymorphism 3066 9407 25 TTT-CTT Phe-Leu polymorphism 3090 9481 26 GTC-GTT Val-Val polymorphism 3110 9541 26 CCT-CCC Pro-Pro polymorphism 3139 9627 27 GGC-TGC Gly-Cys 3180 9751 27 TGG-TGA Trp-X termination 3193 9789 28 CCT-CTT Pro-Leu 3206 9827 28 CAG-TAG Gln-X termination 3219 9867 28 t del = ctt-c{circumflex over ( )}tt Leu-Leu frameshift 3223 9880 28 ACG-ACA Thr-Thr polymorphism 3285 10064 29 GTT-ATT Val-Ile 3311 10143 30 CAT-CGT His-Arg 3341 10234 30 CTT-CTC Leu-Leu polymorphism 3348 10255 30 CGG-CGT Arg-Arg polymorphism 3350 10262 31-34 2kb del frameshift after 3350 3375 10334 31 GTG-ATG Val-Met IVS31 + 25del19 frameshift after 3389 3394 10391 32 CAG-TAG Gln-X termination 34−3′UTR 5.5kb del 3474 10631 34 CAG-TAG Gln-X termination 3509 10737 35 ACG-ATG Thr-Met polymorphism 3510 10739 35 CTG-GTG Leu-Val probable path. 3511 10743 35 GCG-GTG Ala-Val 3513 10748 35 CAG-TAG Gln-X termination 3561 10893 36 AGC-AAC Ser-Asn probable poly. 3579 10947 36 t ins = ttc-tt{circumflex over ( )}t Phe-Phe frameshift 3589 10976 36 CTG-TTG Leu-Leu polymorphism IVS37- intron 37 unknown poly 10C-A 3631 11104 37 GAG-GAC Glu-Asp 3677 11241 38 ATG-ACG Met-Thr 3692 11284 38 t ins = ggc-gg{circumflex over ( )}t Gly-Gly frameshift 3692 11285 38 c ins = tca-{circumflex over ( )}ctc Ser-Leu frameshift 3711 11342 38 CGG-GGG Arg-Gly frameshift 3747 11449 39 15bp del = cgg-{circumflex over ( )}cgg Arg-Arg in frame deletion 3749 11457 39 15bp del = gcg-{circumflex over ( )}cag Arg-Gln in frame deletion 3752 11466 39 CGG-CAG Arg-Gln IVS39 + 1G-C Ggt-Gct splice donor I39E40−25 to 72bp del I39E40 + 47 3370 11521 40 TCG-TCA Ser-Ser polymorphism 3780 11549 40 10bp ins = tac-t{circumflex over ( )}ac Tyr-Tyr frameshift 3781 11554 40 GAC-GAT Asp-Asp polymorphism 3791 11584 40 TCG-TCC Ser-Ser polymorphism 3794 11592 40 TGG-TAG Trp-X termination IVS41- intron 41 unknown poly 11C-T 3818 11665 41 TAC-TAA Tyr-X termination 3820 11669 41 CAG-TAG Gln-X termination 3837 11720 41 CAG-TAG Gln-X termination 3971 12124 43 CGC-CGT Arg-Arg polymorphism 3984 12163 43 TCC-TCG Ser-Ser polymorphism 3985 12165 43 GCA-GGA Ala-Glu 3985 12168 43 GCC-GGG Ala-Gly probable poly. 3991 12184 43 GCC-GCG Ala-Ala polymorphism 12187 43 9bp ins in frame IVS43 + 14del20 complex splicing IVS43 + 17del18 complex splicing 44 CAG-CAC splice acceptor skip exon 44 4010 12239 44 CAG-TAG Gln-X termination 4011 12244 44 TGG-TGA Trp-X termination 4014 12252 44 tt del = ttt-t{circumflex over ( )}gg Phe-Trp frameshift 4017 12262 44 at del = aca-ac{circumflex over ( )}t Thr-Thr frameshift 4020 12269 44 CGA-TGA Arg-X termination 4024 12281 44 GAG-TAG Glu-X termination 4027 12290 44 g ins = ggg-gg{circumflex over ( )}g Gly-Gly frameshift 4031 12303 44 GGC-GAC Gly-Asp 4032 12307 44 CTG-CTC Leu-Leu polymorphism 4039 12328 44 TAC-TAA Tyr-X termination 4041 12332 44 CAG-TAG Gln-X termination 4044 12341 44 ATC-GTC Ile-Val probable poly. 44 GGT-GCT splice donor del of 4001-4045 45 CAG-CAA splice acceptor skip exon 45 4058 12384 45 GCC-GTC Ala-Val probable poly. 4059 12386 45 CAG-TAG Gln-X termination 4069 12416 45 20bp ins = ggg- Gly-** frameshift g{circumflex over ( )}**** 4075 12438 45 20bp ins = gcc-gc{circumflex over ( )}g Ala-Ala frameshift 4086 12469 45 TGT-TGA Cys-X termination 4091 12483 45 GCA-GCG Ala-Ala polymorphism 4101 12511 45 g ins = -ggg-gg{circumflex over ( )}g Gly-Gly frameshift 4124 12581 45 CAG-TAG Gln-X termination 4126 12589 45 TAC-TAG Tyr-X termination 4131 12601 45 gtt del = gagtt-ga{circumflex over ( )}gtt Leu-Phe frameshift 4135 12614 45 AGG-GGG Arg-Gly 4136 12617 45 CTG-TTG Leu-Leu polymorphism 4136 12617 45 c del = ctg-{circumflex over ( )}tgc Leu-Cys frameshift 4139 12628 45 TGG-TGA Trp-X termination 4145 12644 45 GTC-ATC Val-Ile probable poly. IVS45 + intron 45 unknown poly 17insG 4153 12668 46 CGC-TGC Arg-Cys 4168 12714 46 duplication of frameshift 23bp 4176 12739 46 a del = cca-cc{circumflex over ( )}c Pro-Pro frameshift 4189 12777 46 TCC-TTC Ser-Phe polymorphism 4198 12801 46 del 28 frameshift 4209 12838 46 CCT-CCC Pro-Pro polymorphism 4224 12882 46 CAG-CCG Gln-Pro probable path. 4227 12890 46 CGA-TGA Arg-X termination 4236 12919 46 TAC-TAa/g Tyr-X termination 4254 12973 46 CCC-CCT Pro-Pro polymorphism 4275 13034 46 CGG-TGG Arg-Trp probable path.

[0096] III. Identification of Unique Sites within PKD Genes

[0097] Due to the fact that 70% of the PKD-1 gene is replicated as non-functional homologues with more than 95% sequence identity to PKD-1, the identification of PKD-1 unique sites are critical or the development of a genetic testing method. With the successful decoding of human genome sequences, the unique sites within the PKD genes may be identified by comparing genomic DNA sequences comprising a PKD gene with genomic DNA sequences comprising a PKD homologue. Useful databases and computer programs are known in the art (e.g., databases available through NCBI at www.ncbi.nlm.nih.gov; and computer programs available at http://www.ncbi.nlm.nih.gov/BLAST and DNAStar, www.dnastar.com). A unique site refers to a stretch of sequence within a PKD gene which shares less than or equal to 80% (e.g., less than or equal to 70%, or 60%, or 50% or 40% or 30% or 20% or 10%) sequence identity to a PKD homologue or other sequences.

[0098] Several unique sites (e.g., single copy site) have been described in Rossetti et al., 2000, Am. J. Hum. Genet. 68:46-63, the entirety of which hereby incorporated by reference. A novel unique site (5′ AGG TCC AGG GCG ACT CGC TGG 3′, or 5′ CAG GGC CAC ACG CGC TGG GCG 3′, or their complement thereof) is identified for PKD-1 by Applicants of the present application. Other unique sites may be found in, for example, in U.S. Pat. Nos. 6,228,591 and 6,031,088, each of which is incorporated herein by its entirety.

[0099] The identified unique sites can be used for designing PKD-specific primers for the amplification of authentic PKD genes. The length of a unique site may vary from several nucleotides to thousands of nucleotides. Most of unique site identified comprises less than or equal to 100 nucleotides, e.g., less than or equal to 50 nucleotides, or less than or equal to 30 nucleotides. Amplification using PKD-specific primers would increase the specificity of the amplification reaction and reduce the amount by-products amplified from PKD homologues. The specifically amplified product of authentic PKD genes may be subsequently used for sequencing to identify allele variant, e.g., a mutant PKD gene, in an individual or for cloning and/or expression for other analysis.

[0100] IV. PKD-Specific Primers Useful for the Invention

[0101] Samples to be analyzed for the presence or absence of mutations often contain amounts of material too small to detect. The first step in mutation detection assays is, therefore, sample amplification. A preferred amplification reaction of the invention is PCR. PCR amplification comprises steps such as primer design, choice of DNA polymerase enzyme, the number of amplification cycles and concentration of reagents. Each of these steps, as well as other steps involved in the PCR process affects the purity of the amplified product. Although the PCR process and the factors which affect fidelity of replication and product purity are well known in the PCR art, these factors have not been addressed, heretofore, in relation to mutation detection of PKD genes using the separating method of the invention, e.g., DHPLC.

[0102] Any primer which anneals, under specific stringent conditions, to a sequence within an authentic PKD gene, but not to a PKD homologue or other sequences is a useful PKD-specific primer according to the invention. Sequences of the identified unique sites serve as the basis for designing PKD-specific primers useful according to the invention. The primers, according to the subject invention, may be incorporated into a convenient kit for identifying a PKD patient.

[0103] A. Criteria for Selecting Primers

[0104] A PKD species-specific primers preferably comprise a sequence complementary to a sequence located within a unique site of a PKD gene. The PKD-specific primer may be complementary to a unique site of a normal or a mutant PKD gene, so long as the primer preferably anneals to an authentic PKD gene other than a PKD homologue.

[0105] PKD species-specific primers may be selected manually by analyzing sequences of the unique sites identified for a PKD gene. When the sequence of a DNA fragment to be amplified by PCR is known, commercially available software can be used to design primers which will produce either the whole fragment, or any sequence within the fragment. The melting map of a fragment can be constructed using software such as MacMelt® (BioRad Laboratories, Hercules, Calif.), MELT (Lerman et al. Meth. Enzymol. 155:482 (1987)), or WinMelt™ (BioRad Laboratories).

[0106] It is known in the art that primers that are about 18-25 bases long and with 50% G-C content will work well at annealing temperature at about 52-58° C. These properties are preferred when designing primers for the subject invention. Longer primers, or primers with higher G-C contents, have annealing optimums at higher temperatures; similarly, shorter primers, or primers with lower G-C contents, have optimal annealing properties at lower temperatures. A convenient, simplified formula for obtaining a rough estimate of the melting temperature of a primer 17-25 bases long is as follows:

Melting temperature (Tm in °C.)=4×(# of G+# of C)+2×(# of A+# of T)

[0107] The overall design process design consists of both long range (i.e., for the first round PCR) and short range primer (i.e., for the nested PCR) design. In long range primer design, the objective is to design primers that produce good quality PCR products. “Good quality” PCR products are defined herein to mean PCR products produced in high yield and having low amounts of impurities such as primer dimers and PCR induced mutations. Good quality PCR can also be affected by other reaction parameters, such as the enzyme used, the number of PCR cycles, the concentration and type of buffer used, temperature thermal cycling procedures and the quality of the genomic template. Methods for producing good quality PCR products are discussed by Eckert et al. (PCR: A Practical Approach, McPherson, Quirke, and Taylor eds., IRL Press, Oxford, Vol. 1, pp. 225-244, 1991). This reference and the references therein are incorporated herein in their entireties.

[0108] Short range primer design should fulfill two requirements. First, it should fulfill all the requirements of long range primer design and give good quality PCR products. In addition, it must produce fragments that allow the DHPLC method to detect a mutation or polymorphism regardless of the location of the mutation or polymorphism within the amplified fragment. For example, large DNA fragments, having up to several thousand base pairs, can be amplified by PCR. If the only goal of the amplification is to replicate the desired fragment, then there is a large latitude in the design of primers which can be used for this purpose. However, if the purpose of a PCR amplification is to produce a DNA fragment for mutation detection analysis by DHPLC, then primers must be designed such that the fragment produced in the PCR process is capable of being detected, and will produce a signal, when analyzed by DHPLC. In a preferred embodiment of the invention, the length of an amplified product is 150-600 bps. In a more preferred embodiment, the fragment length for DHPLC mutation detection analysis is 150-400 bp.

[0109] There are two goals of designing short range primers. One goal for primer design is if the analysis is used as a “mutation analysis” test. Another goal is in analysis for research or diagnostic purposes, e.g., for identifying a PKD patient. “Mutation analysis” is defined herein as the study or analysis of DNA fragments to determine if the fragments contain variations (i.e., mutations or polymorphisms) in a population and correlate that variation to disease. It is to be understood that, within the context of this invention, the term “mutation” does not include a polymorphism (e.g., normal) which is silent for the disease. When DHPLC is used as a mutation analysis technique, then an important aspect of the present invention is a method for designing primers to produce a fragment in which a putative mutation can be detected, regardless of where the mutation site is located within the fragment. If the mutation is known, on the other hand, then the primer design can be further refined so that the analysis is optimized, i.e., the resolution of the homoduplex and the heteroduplex peaks in DHPLC is maximized. By improving the resolution for the analysis of known mutations, accuracy of analysis can be performed. Improved resolution is required for diagnostic mutation applications. Furthermore, with improved resolution, automatic identification of the positive presence of mutation can be more easily implemented with appropriate software and an algorithm that overlays and comparatively measures the peaks of the normal and mutant DNA samples.

[0110] Another method of primer design for mutation analysis applications is to design the primers so that the region of interest is at a lower melting domain within the fragment. In this case the primers are preferred to be designed so that the fragment being measured will overlap the regions of interest as the analysis is performed traveling down the exon. In these cases, the temperature difference between the higher melting domain and the lower melting domain is preferred to be greater than 5° C. and most preferred to be greater than 10° C.

[0111] Once the mutation of interest is identified, primers can be redesigned for diagnostic or clinical applications. In these cases, the mutation is preferably located within 25% or 25 bases of the end whichever is closer to the end. The other end of the fragment contains a higher melting domain of preferably 5° C., more preferably 10° C. higher, and most preferably 15° C. higher than the lower domain where the mutation is located. If the primer selection does not result in a high melting domain on the opposite end of the fragment, then a G-C clamp can be applied to increase the melting temperature at the desired end (e.g., an A-T rich end) (Myers et al., 1985, Nucleic Acids Res. 13:3111). G-C clamping is a technique in which additional G or C bases are included on the 5′ end of one or both of the primers. The polymerase enzyme will extend over these additional bases incorporating them into the amplified fragment thereby raising the melting temperature of the end(s) of the fragment relative to that in the vicinity of the mutation. For example, in cases where the mutation is in the center of the amplified fragment and the length is less than 100 bp and the melting profile is flat, or in cases where the mutation in a high melting region of the fragment and a higher melting region is in effect a G-C rich region, a G-C clamp may be necessary. In these cases, proper primer selection will result in a fragment in which the mutation can be detected. The size of the G-C clamp can be up to 40 bp and as little as 4 or 5 bp. The most preferred G-C clamp for mutation detection by DHPLC is 10 to 20 bp.

[0112] If it is not possible to design primers which will produce, upon PCR amplification, domains having a constant melting range or domains within a fragment which are sufficiently close in Tm, then it may be necessary to lower the Tm of a domain of interest for successful mutation detection by DHPLC. This can be done, for example, by substituting dGTP with the analog 7-deaza-2′-dGTP which is known to effectively lower the melting temperature of G-C base pairs (Dierick et al.,1993, Nucl. Acids Res. 21:4427). If it is necessary to raise the Tm of the domain, then 2, 6-aminopurine can be used in place of dGTP in the PCR amplification.

[0113] In a most preferred embodiment, the primers are selected so that the mutation is located in a “lower melting” domain of the fragment. However, a mutation can also be detected by DHPLC in a high melting domain of the fragment either if the high melting domain does not have a melting temperature that is too different from other domains in the fragment or if a higher column temperature is used that is optimized for the higher melting domain of the fragment.

[0114] The long range primer design described above can be further refined by local primer design in which several other factors should be considered. For example, primers with non-template tails, such as universal sequencing primers or T7 promoters, may need to be avoided. The preferred primer has a Tm of about 56° C. The difference in Tm between the forward and reverse primers is preferably about 1° C. The difference in Tm between primer and template is preferably 25° C. The 3′-pentomer of each primer is preferably be more stable than ΔG°=−6 kcal/mol (i.e., more negative). Any possible primer dimers are preferably be less stable than the 3′-pentomer by at least 5 kcal/mol (i.e., 5 kcal more positive). Any primer self annealing loops are preferably to have a Tm of less than 12° C. Primers are preferably be of high purity without failure sequences. To avoid degradation, storage in Tris-HCl (pH 8.0) buffer is preferable to pure water.

[0115] In some embodiments, it is more convenient to directly separate a long fragment, e.g., an exon, of up to 5 kb (e.g., up to 4 kb, or up to 3kb, or up to 2 kb, or up to 1 kb) for mutations. Such long fragments generally contain multiple melting temperature domains. Double-stranded DNA fragments melt in a series of discontinuous steps as different regions with differing thermal stabilities which denature in response to increasing temperature. These different regions of thermal stability are referred to as “domains”, and each domain is approximately 50-300 bp in length. Each domain has its own respective Tm and will exhibit thermodynamic behavior which is related to its respective Tm. The presence of a base mismatch within a domain will destabilize it, resulting in a decrease in the Tm of that domain in the heteroduplex relative to its fully hydrogen-bonded counterpart found in the homoduplex. Generally the presence of a base mismatch will lower the Tm by approximately 1-2° C.

[0116] In accordance with the preferred embodiments, optimal results have been obtained using primers which are 18-51 in length and DNA sequence to the primers with SEQ ID NOs. 3-49 (Table 3 and Table 4). However, one skilled in the art will recognize that the length of the primers used may vary. For example, it is envisioned that shorter primers containing at least 15, and preferably at least 17, consecutive bases of the nucleotide sequences of these primers SEQ ID NOs. 3-49 may be suitable. The exact upper limit of the length of the primers is not critical. However, typically the primers will be less than or equal to approximately 60 bases, preferably less than or equal to 50 bases. Further still, the bases included in the primers may be modified as is conventional in the art, including but not limited to, incorporating detectable labels such as biotin, or fluorescent labels. TABLE 3 Examples of useful pkd-1 specific primers* SEQ ID NO. Primer Name Primer Sequence 1X1F 5′ CGT CGC TCA GCA GCA GGT CG 3′ 1X1R 5′ CGT CCT GCT TCC CGT CCC G 3′ 1X2F 5′ GCG GCC CGC CGC CCC CGC CGT TGG GGA TGC TGG CAA TGT GTG 3′ 1X2R 5′ GGG ATT CGG CAA AGC TGA TG 3′ 1X3F 5′ TTC CAT CAG CTT TGC CGA AT 3′ 1X3R 5′ ATC TGG TCT CAA GCC TGG AAG 3′ 1X4F 5′ GCC CCG CGC CCG TCC CGC CGC CCC CGC CGA GAC CCT TCC CAC CAG ACC T 3′ 1X4R 5′ CGC CCC CGC CCG TGA GCC CTG CCC AGT GTC T 3′ 1X5AF 5′ GCG GCC CGC CGC CCC CGC CGG AGC CAG GAG GAG CAG AAC CC 3′ 1X5AR 5′ CAG AGG GAC AGG CAG GCA AAG G 3′ 1X5BF 5′ GCC CCC GCC GCC CAG CCC TCC AGT GCC T 3′ 1X5BR 5′ ATC GCT ATG TGC TGC CTG GG 3′ 1X5CF 5′ CCG AGG TGG ATG CCG CTG 3′ 1X5CR 5′ GAA GGG GAG TGG GCA GCA GAC 3′ 1X6F 5′ CAC TGA CCG TTG ACA CCC TCG 3′ 1X6R 5′ TGC CCC AGT GCT TCA GAG ATC 3′ 1X7F 5′ GGA GTG CCC TGA GCC CCC T 3′ 1X7R 5′ CCC CTA ACC ACA GCC AGC G 3′ 1X8F 5′ TCT GTT CGT CCT GGT GTC CTG 3′ 1X8R 5′ GCA GGA GGG CAG GTT GTA GAA 3′ 1X9F 5′ GCG GCC CGC CGC CCC CGC CGG GTA GGG GGA GTC TGG GCT T 3′ 1X9R 5′ GAG GCC ACC CCG AGT CC 3′ 1X10F 5′ GTT GGG CAT CTC TGA CGG TG 3′ 1X10R 5′ CGC CGC CCC CGC CCG GGA AGG TGG CCT GAG GAG AT 3′ 1X11AF 5′ GCG GCC CGC CGC CCC CGC CGG GGG TCC ACG GGC CAT G 3′ 1X11AR 5′ AAG CCC AGC AGC ACG GTG AG 3′ 1X11BF 5′ CCG CCG CCC CCG CCG CTG CCC TGC CTG TGC CCT G 3′ 1X11BR 5′ GCC CCG CGC CCG TCC CGC CGC CCC CGC CCG TTC CAC CAC CAC GTC CAC CAC 3′ 1X11CF 5′ GTG GTG GAC GTG GTG GTG GAA 3′ 1X11CR 5′ GGC TGC TGC CCT CAC TGG GAA 3′ 1X12F 5′ TAA GGG CAG AGT CCT CCA CAG 3′ 1X12R 5′ CCA CCC CCG CCC ACC TAC TGA G 3′ 1X13F 5′ GCG GCC CGC CGC CCC CGC CGT GGA GGG AGG GAC GCC AAT C 3′ 1X13R 5′ GAG GCT GGG GCT GGG ACA A 3′ 1X14F 5′ CCC GGT TCA CTC ACT GCG 3′ 1X14R 5′ CCC CCG CCC GCC GTG CTC AGA GCC TGA AAG 3′ 1X15AF 5′ GGC GGG GGG CTT CTG CCG AGC GGG TGG GGA GCA GGT GG 3′ 1X15AR 5′ CGC CGC CCC CGC CCG GCT CTG GGT CAG GAC AGG GGA 3′ 1X15BF 5′ CGC CTG GGG GTG TTC TTT 3′ 1X15BR 5′ ACG TGA TGT TGT CGC CCG 3′ 1X15CF 5′ GCC CCC GCC GGG GCG CCC CCG TGG TGG TCA GC 3′ 1X15CR 5′ CAG GCT GCG TGG GGA TGC 3′ 1X15DF 5′ CTG GAG GTG CTG CGC GTT 3′ 1X15DR 5′ CGC CCC CGC CCG CTG GCT CCA CGC AGA TGC 3′ 1X15EF 5′ CGT GAA CAG GGC GCA TTA 3′ 1X15ER 5′ CCC CCG CCC GGC AGC AGA GAT GTT GTT GGA C 3′ 1X15FF 5′ CCG CCG CCC CCG CCG CCA GGC TCC TAT CTT GTG ACA 3′ 1X15FR 5′ TGA AGT CAC CTG TGC TGT TGT 3′ 1X15GF 5′ CTA CCT GTG GGA TCT GGG G 3′ 1X15GR 5′ TGC TGA AGC TCA CGC TCC 3′ 1X15HF 5′ GGG CTC GTC GTC AAT GCA AG 3′ 1X15HR 5′ CGC CGC CCC CGC CCG CCG CCC ACC ACC TGC AGC CCC TCT A 3′ 1X15IF 5′ GCG GCC CGC CGC CCC CGC CGC CGC CCA GGA CAG CAT CTT C 3′ 1X15IR 5′ CGC TGC CCA GCA TGT TGG 3′ 1X15JF 5′ GGC CGG CAG CGG CAA AGG CTT CTC 3′ 1X15JR 5′ GCC CAG CAC CAG CTC ACA T 3′ 1X15KF 5′ CGA GCC ATT TAC CAC CCA TAG 3′ 1X15KR 5′ GGC AGC CAG CAG GAT CTG AA 3′ 1X15LF 5′ CTG TGG GCC AGC AGC AAG GTG 3′ 1X15LR 5′ CCT GAA CCT CCA GCA CCA GCG 3′ 1X15MF 5′ AGG TCC AGG GCG ACT CGC TGG 3′ 1X15MR 5′ CAG GGC CAC ACG CGC TGG GCG 3′ 1X15NF 5′ TTG GAG GCC CAC GTT GAC CTG 3′ 1X15NR 5′ CCC CCG CCC GCA TGG GTG TGG ACG GGT GAG G 3′ 1X16F 5′ TAA AAC TGG ATG GGG CTC TC 3′ 1X16R 5′ GGC CTC CAC CAG CAC TAA 3′ 1X17F 5′ GGG TCC CCC AGT CCT TCC AG 3′ 1X17R 5′ TCC CCA GCC CGC CCA CA 3′ 1X18F 5′ GCC CCC TCA CCA CCC CTT CT 3′ 1X18R 5′ TCC CGC TGC TCC CCC CAC GCA 3′ 1X19F 5′ GAT GCC GTG GGG ACC GTC 3′ 1X19R 5′ GTG AGC AGG TGG CAG TCT CG 3′ 1X20F 5′ CCA CCC CCT CTG CTC GTA GGT 3′ 1X20R 5′ GGT CCC AAG CAC GCA TGC A 3′ 1X21F 5′ TGC CGG CCT CCT GCG CTG CTG A 3′ 1X21R 5′ GCG GGC AGG GTG AGC AGG TGG GGC CAT CC 3′ 1X22F 5′ GAG GCT GTG GGG GTC CAG TCA AGT GG 3′ 1X22R 5′ AGG GAG GCA GAG GAA AGG GCC GAA C 3′ 1X23AF 5′ CGT CCC GCC TGC ACT GAC CTC ACG CAT GT 3′ 1X23AR 5′ CGG CCC GCC GCC CCC GCC CGG CCA AAG GGA AAG GGA TTG GA 3′ 1X23BF 5′ CCG CGG AGC CTG CTG TGC TAT 3′ 1X23BR 5′ CCG CCG CCC CCG CCC GCT TGG TGG AGA CGG TGT AGT TGC 3′ 1X23CF 5′ TCC AAT CCC TTT CCC TTT GGC 3′ 1X23CR 5′ CAG CAG CCC ATG AAA CAG AAA G 3′ 1X24F 5′ TAT GCT TTC AGG CCC GTG GCA 3′ 1X24R 5′ AGA GCC CAT ACC CGG TCC AGT CC 3′ 1X25F 5′ GGA CTG GAC CGG GTA TGG GCT CT 3′ 1X25R 5′ CCC CCG CCC GCA CCC AGG CCC TCC TCG ACT C 3′ 1X26F 5′ CCC CCG CCG CTG GGT GGG CTC GGC TCT ATC 3′ 1X26R 5′ TGG TAG CGA TGC TCA CGT CAC TT 3′ 1X27F 5′ CAG GCC AAA GCT GAG ATG ACT TG 3′ 1X27R 5′ AGA GGC GCA GGA GGG AGG TC 3′ 1X28F 5′ CCC TCT GCC CCC GCA TTG 3′ 1X28R 5′ AAG CGC AAA AGG GCT GCG TCG 3′ 1X29F 5′ GGC CCT CCC TGC CTT CTA GGC G 3 1X29R 5′ CCG TGC TGT GTG GAG GAG AG 3′ 1X30F 5′ CCT CTT CCT GCC CAG CCC TTC 3′ 1X30R 5′ CTT CCC GAG CAG CCT TTG GTG 3′ 1X31F 5′ CTG AGC TGC CGC CCG CTG AC 3′ 1X31R 5′ AGG ACC CCC AGC CCA GCC CA 3′ 1X32F 5′ CTT GGC GCA GCT TGG ACT 3′ 1X32R 5′ ACA CCC AGC AAG GAC ACG CA 3′ 1X33F 5′ TGT GAC ACA TCC CCT GGT AC 3′ 1X33R 5′ GCA AGG GTG AGC TTC AGA GC 3′ 1X34F 5′ GCC CCG CGC CCG TCC CGC CGC CCC CGC CCG ACC CTA TGC CTC CTG TAC CTC 3′ 1X34R 5′ CCC CTC CTC TGG CAA TCC 3′ 3 1X35F 5′ TGG CTG CAA CTG CCT CCT GG 3′ 4 1X35R 5′ AAG CAG AGA CAG ACC TGT GAG AG 3′ 5 1X36F 5′ GCC CCC GCC GCT CTC ACA GGT CTG TCT CTG CTT C 3′ 6 1X36R 5′ GGC CTG TAG CCT ACC CCT GG 3′ 7 1X37F 5′ GGA CCC CTC TGA AGC CAC C 3′ 8 1X37R 5′ GGG AGG TGG GAG ACA AGA GAC 3′ 9 1X38F 5′ AAA GCC CTG CTG TCA CTG TGG 3′ 10 1X38R 5′ AAC TAA AGC CCA GAA GAC AGA CC 3′ 11 1X39F 5′ AAC TGT CTG CCC CAG AAC ATC 3′ 12 1X39R 5′ CTA AAG GCT GCT CTC TCA ACA AG 3′ 13 1X40F 5′ ACT CCT GTT GGG TTT TGA TGA G 3′ 14 1X40R 5′ GAG AAC TAC TCC CTT GTC CTT GG 3′ 15 1X41F 5′ ACG CCA AGG ACA AGG GAG TAG TTC 3′ 16 1X41R 5′ TGG GCT CCT GGC TGG TGA CTG C 3′ 17 1X42F 5′ GCG GCC CGC CGC CCC CGC CGC TAC TGA CCC GCA CCC TCT G 3′ 18 1X42R 5′ GCT GCG AGG GGT GAG ACG 3′ 19 1X43F 5′ GCG GCC CGC CGC CCC CGC CGC GTC CCT CCC GCC CTC CTG ACC 3′ 20 1X43R 5′ GCC CCC GCC GCT GCG GAC GAG AAA TCT GTC TGC TTG 3′ 21 1X44F 5′ CAG GGC TGC AAG CAG ACA GA 3′ 22 1X44R 5′ CTG AGC TAA GAC GCC CTC CC 3′ 23 1X45F 5′ CTG TAC GCC CTC ACT GGT GTC 3′ 24 1X45R 5′ GGC ACA GGG GCT CAG TCA GTC 3′ 25 1X46AF 5′ GGA CTG ACT GAG CCC CTG TGC 3′ 26 1X46AR 5′ AGT CGG TCA AAC TGG GTG AG 3′ 27 1X46BF 5′ CAA GGT GTG AGC CTG AGC CC 3′ 28 1X46BR 5′ CGG TGT CCA CTC CGA CTC CAC 3′ # (1X15 A F). The last letter indicates the direction of the primer as either forward or reverse (1X15A F ).

[0117] TABLE 4 Examples of useful pkd-2 specific primers* SEQ ID NO. Primer Name Primer Sequence 29 2X1AF 5′ CCG CCC CCG CCG CGC GCC GGA CGC CAG TGA CC 3′ 2X1AR 5′ CCT GCC GGG AGC ACG ACG AG 3′ 30 2X1BF 5′ GCC CCC GCC GCC GCG GCC TCC CCT TCT CCT 3′ 2XIBR 5′ CTG GGC TGG GGC ACG GCG GG 3′ 2X1CF 5′ GGG GGC TAC CAC GGC GCG GGC 3′ 31 2X1CR 5′ CGG CCC GCC GCC CCC GCC CGC GGC CGT T GGT TCG TGC ATC TG 3′ 32 2X2F 5′ GCC CCC GCC GAA ATG ATA TCT TTT TTC TTC A 3′ 33 2X2R 5′ CCC CCG CCC GAA CTT TCC CAT TAG TGC A 3′ 2X3F 5′ TTG GGG CGT TCA TTT GGA TC 3′ 34 2X3R 5′ CGC CGC CCC CGC CCG TGT GAT AGA GAG CTT TCA 3′ 35 2X4F 5′ CCG CCG CCC CCG CCG CTT TTT CAA AGA T TTC CTT TGC 3′ 36 2X4R 5′ TAT CAC CGA GTG CCA ATG AG 3′ 37 2X5F 5′ CCG CCG CCC CCG CCG GCC TCA AGT GTT C CTG AT 3′ 2X5R 5′ ACC ACA CAG AAA TAG GAG GG 3′ 2X6F 5′ TTG TTA TTG TTT TAA TTG TTC TTA 3′ 38 2X6R 5′ CCC CCG CCC GTT GTA GAA TAG AAT AGG TTT GG 3′ 39 2X7F 5′ GCC CCC GCC GTT GGT GAA GAA AAA TAT AGT CA 3′ 40 2X7R 5′ CGC CGC CCC CGC CCG TGG AAC TCA TTT T TTA AAG A 3′ 41 2X8F 5′ GCG GGG GCG GCG GGC CGT TTT ATT ATA AGT CAC ACC 3′ 2X8R 5′ CTA CTC TGA CTA AAT TTT TCT TCT T 3′ 2X9F 5′ TTT GGT TTT GTA TTG TGG TG 3′ 2X9R 5′ AAG GAT TTA CGA AGT TTA AAT TG 3′ 42 2X10F 5′ GCC CCC GCC GCT TCC TTT AAT TTT TGC CC 3′ 43 2X10R 5′ CGC CGC CCC CGC CCG GAA ACA ATG CTC A TTA TGT CAG 3′ 44 2X11F 5′ CCG CCG CCC CCG CCG AAA CCA AGT CTT T TTT TTT CTC 3′ 2X11R 5′ AGA ACC TCA GGA AGC ATG ATT 3′ 45 2X12F 5′ CCG CCG CCC CCG CCG GAT GAA TGT TAT C TAT CCT CTC 3′ 2X12R 5′ TAG GTA CCA AAT CAA ATC CG 3′ 2X13F 5′ GTC TCA GTG TTC TGC TCC TC 3′ 46 2X13R 5′ CGC CGC CCC CGC CCG GCA AAT TCT GCC A TCC TTT A 3′ 47 2X14F 5′ GCC CCC GCC GTT TGT CCC TCT GTA CTG TG 3′ 2X14R 5′ AAA TAC AAC TGT CAG CAA CAT A 3′ 48 2X15F 5′ CCG CCC CCG CCG TGA CCC CCA ACA CCA G TC 3′ 49 2X15R 5′ CGG CCC GCC GCC CCC GCC CGG GAC AGC C TTC CTC ACT T 3′ # (2X15 R ).

[0118] B. Primer Combinations Useful for PKD-specific Amplification

[0119] The specifically amplified product can be generated by using one or more PKD-specific primers. Preferably, both primers used to generate one amplified product are PKD-specific primers. However, one PKD-specific primer can be used in combination with another non PKD-specific primer which is not complementary to a unique site of a PKD gene. The non PKD-specific primer is preferably designed according to the same criteria described above herein for the PKD-specific primers and is preferably to be completely complementary to a sequence other then a unique sequence in a PKD gene. A non PKD-specific primer may also be used as a control primer included in the amplification reaction to generate a control product.

[0120] Optimal results may be obtained by using one forward and one reverse primer listed in Table 4 and Table 5, although other combinations may also be used. In a preferred embodiment, a primer pair is selected so that the length of an amplified product is 150-600 bps. In the most preferred embodiment, a primer pair is selected so that the amplified fragment length for DHPLC mutation detection analysis is 150-400 bp.

[0121] C. Primer Synthesis

[0122] Methods for synthesizing primers are available in the art. The oligonucleotide primers of this invention may be prepared using any conventional DNA synthesis method, such as, phosphotriester methods such as described by Narang et al. (1979, Meth. Enzymol., 68:90) or Itakura (U.S. Pat. No. 4,356,270), or and phosphodiester methods such as described by Brown et al. (1979, Meth. Enzymol., 68:109), or automated embodiments thereof, as described by Mullis et al. (U.S. Pat. No. 4,683,202). Also see particularly Sambrook et al.(1989), Molecular Cloning: A Laboratory Manual (2d ed.; Cold Spring Harbor Laboratory: Plainview, N.Y.), herein incorporated by reference.

[0123] V. Preparing Template for Amplification Reaction

[0124] Any sample comprising a nucleic acid comprising the entire or a portion of SEQ ID NO. 1 or 2 or their variants (e.g., polymorphism forms or mutant forms) may be used to as template for amplification reaction of the present invention. Useful templates, according to the invention, include, but are not limited to, genomic DNA preparation, total RNA preparation, crude cell lysate and tissue sample.

[0125] It's preferred to use genomic DNA as template for PKD-specific amplification of the subject invention. While it is envisioned that crude cell lysate or tissue sample may be used, one skilled in the art will recognize that any non-DNA material present in the sample may interfere with the polymerase reaction or subsequent analysis.

[0126] Genomic DNA can be isolated from tissue samples or cells. Preferably, the genomic DNA used as template for the invention is isolated under conditions which preclude degradation and contamination. Tissue samples or cells may be digested with a protease so that there is likely to be little or no DNAase activity. The digest is extracted with a DNA solvent. The extracted genomic DNA may be purified by, for example, dialysis or chromatography. Suitable genomic DNA isolation techniques are known in the art, for example, as described in Current protocols in molecular biology, Ausubel et al., John Weley & Sons, Inc., 1997.

[0127] Preferably, genomic DNA or cDNA is extracted from cell lysate of tissue samples taken from an individual and used as template for PKD amplification. Collecting a tissue sample also includes in vitro harvest of cultured human cells derived from an individual's tissue or any means of in vivo sampling directly from a subject, for example, by blood draw, spinal tap, tissue smear or tissue biopsy. Optionally, tissue samples are stored before analysis by well known storage means that will preserve a sample's nucleic acids in an analyzable condition, such as quick freezing, or a controlled freezing regime, in the presence of a cryoprotectant, for example, dimethyl sulfoxide (DMSO), glycerol, or propanediol-sucrose. Tissue samples can also be pooled before or after storage for purposes of amplifying them for analysis. In some embodiments, the sample contains DNA, tissue or cells from two or more different individuals.

[0128] Any human tissue containing nucleic acids can be sampled and collected for the purpose of practicing the methods of the present invention. A most preferred and convenient tissue for collecting is blood. No patient preparation is necessary prior to blood draw. No medications are known to interfere with sample collection or testing. Usual aseptic techniques and avoidance of contamination are necessary.

[0129] Preferably, DNAs are extracted from blood on the day it was drawn. Blood is preferred to stored at room temperature (72° F. or 25° C.) before use. However, whole blood may be stored for short periods at 4° C. but room temperature is recommended. Whole blood specimens may be stable for 48 hrs. After this time hemolysis may compromise DNA recovery and integrity. The optimal amount of blood for DNA extraction for the PCR assay is preferred to be more than 5 ml, e.g., more than 10.0 ml.

[0130] VI. PCR Amplification Using PKD-specific Primers

[0131] The subject invention provides a method of mutation analysis of a target nucleic acid comprising SEQ ID NO. 1 or 2 or their variants by amplifying the DNA from a sample comprising the target nucleic acid in a polymerase chain reaction and detecting in a specifically amplified product the presence or absence of a mutation in the target nucleic acid.

[0132] Amplification may be carried out by means well known in the art, for example, polymerase chain reaction (PCR), transcription based amplification (reverse transcription), strand displacement amplification (see Current Protocol in Molecular Biology). Preferably, the amplification is carried out by PCR, such as described by Mullis (U.S. Pat. No. 4,683,202), the contents of which are incorporated by reference herein.

[0133] PCR makes possible the amplification (replication) of minute samples of DNA or other nucleic acids of any base pair length (size) by taking advantage of highly selective enzymes called DNA polymerases, to extend small DNA strands called “primers” along a “template”. The minute DNA sample serves as the template. PCR reproduces the complementary sequence of deoxynucleotide triphosphate (dNTP) bases present in the template or any chosen portion thereof. The PCR is commonly used in conjunction with diagnostic techniques where, for example, a DNA sample having a concentration below the limit of detection is amplified by the PCR process, and the larger amount so obtained is subsequently analyzed.

[0134] Apparatus for performing PCR amplifications, e.g. Air Thermo Cycler (Idaho Technologies) and reagents are commercially available from numerous sources, e.g. Perkin-Elmer Catalog “PCR Systems, Reagents and Consumables” (Perkin-Elmer Applied Biosystems, Foster City, Calif.).

[0135] PCR is typically run in a buffer at pH 5-8. The buffer contains a double stranded DNA sample to be amplified, a forward primer, a reverse primer, magnesium (e.g., as MgCl₂), and the four deoxynucleotide triphosphates (dATP, dTTP, dCTP, and dGTP) generally referred to as “dNTPs”, the building blocks of DNA. The reaction mixture is heated to a temperature (e.g., >90° C.) sufficient to denature the DNA sample, thereby separating its two complimentary nucleic acid strands. Alternatively, the DNA may be denatured enzymatically at ambient temperature using a helicase enzyme. If denaturing is effected by heat and a thermostable DNA polymerase is used, the DNA polymerase is added before the reaction is started. Other denaturing conditions are well known to those skilled in the art and are described in U.S. Pat. No. 5,698,400. DNA polymerases are commercially available from a variety of sources, e.g. Perkin-Elmer Applied Biosystems, (Foster City, Calif.) and Stratagene (La Jolla, Calif.).

[0136] The primer sequence is designed to be complimentary to an identified portion of the denatured DNA strands to be replicated by PCR. Upon cooling the reaction to an appropriate annealing temperature, each of the primers anneals to its complimentary base sequence in each strand of the denatured DNA sample to be replicated. Heated to about 70° C. in the presence of the DNA polymerase, the 4 dNTPs and Mg²⁺, replication extends the primers from their 3′-ends by adding complimentary dNTPs along the length of the strand. dNTPs are commercially available from a variety of sources, e.g. Pharmacia (Piscataway, N.J.). By repeating this process numerous times, a geometric increase in the number of desired DNA strands is achieved in the initial stages of the process or as long as a sufficient excess of reagents are present in the reaction medium. Thus, the amount of the original DNA sample is amplified.

[0137] The amount of polymerase must be sufficient to promote DNA synthesis throughout the predetermined number of amplification cycles. Guidelines as to the actual amount of polymerase are generally provided by the supplier of the PCR reagents and are otherwise readily determinable by a person of ordinary skill in the art. Preferably, a DNA polymerase with proof-reading activity is used.

[0138] The amount of each primer must be in substantial excess of the amount of target DNA to be amplified. The amount of primer needed for the reaction mixture can be estimated by one skilled in the art in terms of the ultimate number of amplified fragments desired at the conclusion of the reaction.

[0139] To prevent false positive results, one skilled in the art will recognize that the assays should include negative controls as is conventional in the art. For instance, suitable negative controls may contain no primer or no DNA (i.e. “water controls”). To prevent false negative results, positive controls are provided by the control primers (see below).

[0140] A. Optimization of PCR Conditions

[0141] Successful specific amplification, e.g., an amplification which produces maximal amount of specifically amplified products and minimal amount of non-specifically amplified products, according to the invention, depends in great measure on the specific annealing of the PKD-specific primers to the corresponding matched template. If the primer anneals non-specifically to many different sequences in the reaction mixture, the amplification process will not be specific. Although it is unlikely in most of the embodiments to avoid any non-specific annealing or non-specific amplification, it is desirable to optimize the PCR amplification reaction condition so to reduce the non-specific amplification while increase the specific amplification.

[0142] In addition, PCR induced mutations, wherein a non-complimentary base is added to a template, are often formed during sample amplification. Such PCR induced mutations make mutation detection results ambiguous, since it may not be clear if a detected mutation was present in the sample or was produced during the PCR process. Applicants have recognized the importance of optimizing PCR sample amplification in order to minimize the formation of PCR induced mutations and ensure an accurate and unambiguous analysis of putative mutation containing samples.

[0143] B. Controlling the Specificity of PKD-specific Annealing of PKD-specific Primers.

[0144] The degree of fidelity of replication of DNA fragments by PCR depends on many factors which have long been recognized in the art. Some of these factors are interrelated in the sense that a change in the PCR product profile caused by an increase or decrease in the quantity or concentration of one factor can be offset, or even reversed by a change in a different factor. For example, an increase in the enzyme concentration may reduce the fidelity of replication, while a decrease in the reaction temperature may increase the replication fidelity. An increase in magnesium ion concentration or dNTP concentration may result in an increased rate of reaction which may have the effect of reducing PCR fidelity. A detailed discussion of the factors contributing to PCR fidelity is presented by Eckert et al., (in PCR: A Practical Approach, 1991, McPherson, Quirke, and Taylor eds., IRL Press, Oxford, Vol. 1, pp. 225-244); and Andre, et. al., (1977, GENOME RESEARCH, Cold Spring Harbor Laboratory Press, pp. 843-852). These references and the references cited therein are incorporated in their entirety herein. Thus, availability of a product profile of the PCR process, makes possible the optimization of PCR conditions to improve results in a highly efficient manner.

[0145] In PCR amplification, the specificity of the annealing is most important in the first few cycles. The remaining cycles only serve to expend the pool of template which is amplified in the first few cycles. The specificity of primer annealing to template is controlled by the ionic strength (primarily the K⁺ concentration) of the buffer, the Mg²⁺ concentration (which is bound to dNTPs and therefore affected by the amount of dNTPs), and the annealing temperature of each cycle of the amplification. In preferred embodiments, the dNTP concentrations are 50 nM, preferably 100 nM, more preferably 200 nM.

[0146] Conditions for specific annealing of primers to particular template targets must be determined empirically, usually by varying the annealing temperature in several degree increments and comparing the specificity and sensitivity of the amplification process by agarose gel electrophoresis (See Current Protocol in Molecular Biology, supra).

[0147] Because a unique region to which a PKD-specific primer complement to may differ from a homologue sequence only by a few nucleotides, sometimes by only one nucleotide, the specificity of the amplification reaction needs to be tested for each PKD-specific primer used in the reaction.

[0148] The formula for calculating primer annealing temperature provided above is only a rough guide, successive trials at different annealing temperatures is the usual way to optimize this important parameter in the PKD-specific amplification reaction. Apparatus are available for simultaneous testing of different annealing temperatures of particular primer-template pairs, which enables the optimal annealing temperature to be determined rapidly and reliably (e.g., Robocycler Gradient Temperature Cycler, Cat # 400864, Stratagene; Eppendorf mastercycler gradient, Cat # 5331 000.045, Brinkmann Instruments, Inc. Westbury, N.Y.).

[0149] In some embodiments, the target sequences are amplified at an annealing and extending temperature that is between 1° C. and 10° C. higher than the Tm for the primer pair. Although amplification at this temperature is inefficient, any primer extension that occurs is target specific. Consequently, during the high temperature cycle(s), the sample is enriched for the particular target sequence and any number of cycles, i.e., 1-15 enhances product specificity. The annealing temperature may be then decreased to increase amplification efficiency and provide a detectable amount of PCR product. Or a nested amplification reaction may be performed using the amplified product from the first PCR reaction as template (see below).

[0150] Alternatively, one can simultaneously run a set of reactions at a constant temperature but vary the concentration of KCl or MgCl₂ or add variable amounts of a denaturant such as formamide (e.g., 0, 2, 4, 6%), DMSO (1-10%) to define the optimum conditions for generating a high yield of specific product with a minimum of nonspecific products.

[0151] In one embodiment, a pair of primers comprising at least one selected from the group consisting of SEQ ID NOs. 3-49 is used in the amplification reaction mixture. The orientation of the two primers is opposite to allow the generation of one or more specifically amplified product.

[0152] In some embodiments of the invention, when primers used for PKD-specific amplification are selected from SEQ ID NOs. 3-49, AmpliTaq Gold DNA polymerase with GeneAmp PCR buffer II and MgCl₂ solution and rTth DNA polymerase XL & XL buffer II pack from Perkin Elmer, and TaqPlus Precision PCR system from Stratagene were used. PFUTurbo™ is another high fidelity DNA polymerase having greater proof reading provided by Stratagene.

[0153] In other embodiments, an annealing temperature of above 65° C. (e.g., 68-72° C.) is used for PKD-specific amplification using primers selected from SEQ ID NOs. 3-49.

[0154] In general, it is preferred but not essential that the DNA polymerase is added to the amplification reaction mixture after both the primer and template are added. Alternatively, for example, the enzyme and primer are added last or the reaction buffer or template plus buffer are added last. It is generally desirable that at least one component that is essential for polymerization not be present until such time as the primer and template are both present, and the enzyme can bind to and extend the desired primer/template substrate. This method, termed “hot start,” minimizes the formation of “primer-dimer” and improves specificity of the amplification.

[0155] The degree of specificity of DNA polymerases varies with the reaction conditions employed as well as with the type of enzyme used. No enzyme affords completely error free extension of a primer. Therefore, a non-complimentary base may be introduced from time to time. Such enzyme related errors produce double stranded DNA products which are not exact copies of the original DNA sample, but contain PCR induced mutations. Other PCR process features, such as reaction temperature, primer annealing temperature, enzyme concentration, dNTP concentration, Mg²⁺ concentration, and combinations thereof, all have the potential to contribute to the degradation of the accuracy or fidelity of DNA replication by the PCR process, as described above herein.

[0156] C. Sensitivity of PKD-specific Amplification

[0157] The sensitivity of the PKD-specific amplification of the subject invention depends on the template and primers used in an amplification reaction, as well as ionic strength and annealing temperature of each cycle of the amplification.

[0158] When genomic DNA is used as template, as few as one or two copies of the template (about 3-5 pg) can be used for successful PCR amplification if the reaction condition has been optimized. However, it's known in the art that a higher template concentration may increase the specificity and efficiency of the amplification.

[0159] Shorter fragments are amplified more efficiently than longer fragments. Preferably, primers which generate an amplified product of less than 1 kb, more preferably less than 600 bp, or less than 450 bp in length are used to increase sensitivity of the amplification assay.

[0160] Preferably, the sensitivity of the amplification assay is less than 100 ng genomic DNA template. More preferably, the sensitivity of the assay is less than 10 ng genomic DNA template. More preferably, the sensitivity of the assay is less than 1 ng genomic DNA template. More preferably, the sensitivity of the assay is less than 0.1 ng genomic DNA template. Even more preferably, the sensitivity of the assay is less than 0.01 ng genomic DNA template.

[0161] D. Nested Amplification

[0162] In some embodiments of the invention, a nested amplification is performed using amplified products in a preceding amplification reaction as templates. Preferably, the nested amplification reaction is a nested PCR using PCR amplified products from a preceding PCR reaction as templates. In addition to optimizing the annealing temperature of the primers, “nested” amplification can be used to increase the specificity and sensitivity of the PKD-specific amplification assay.

[0163] For example, a method comprising a nested PCR involves two sequential PCR reactions. After multiple cycles of PCR (e.g., 10 to 40, or 10 to 30 or 10 to 20 cycles) with the first pair of primers comprising at least one PKD-specific primer (e.g., a PKD-specific primer and a control primer or two PKD-specific primers), a small amount aliquot of the first reaction (e.g., 1 μl of a 50 μl reaction) serves as the template for a second multiple cycles of PCR reaction (e.g., 10 to 40, or 10 to 30 or 10 to 20 cycles) with a new set of primers comprising at least one PKD-specific primer (e.g., a PKD-specific primer and a control primer or two PKD-specific primers) that anneal to sequences internal to, or nested between, the first pair.

[0164] Methods for designing nested primers and for performing nested PCR are known in the art (see Current Protocol in Molecular Biology, supra). The general criteria for selecting primers as described above also applies to the design of nested primers. Both nested primers need to anneal to sequences internal to (e.g., within) the first pair of primers and at least one of the nested primers, however, according to the subject invention, needs to be PKD-specific.

[0165] Using the nested PCR procedure, the template that is successfully amplified is selected twice for PKD-specificity. The use of nested PCR can also greatly enhance the yield of the species-specific product, therefore the sensitivity of the assay, when a single primer pair fails by itself.

[0166] A sample comprising genomic DNA or cDNA may be used to provide DNA template for the amplification reaction. Preferably, genomic DNA is used as template. When a sample comprising genomic DNA is used in the reaction mixture, a pair of primers comprising at least one selected from the group consisting of SEQ ID NOs. 3-49 generate at least two specifically amplified product, one from each PKD allele in the genomic DNA sample.

[0167] E. Amplification Controls

[0168] Control primers can be used to serve as positive control for the PKD-specific amplification. The control primers may be added to the same reaction mixture for PKD-specific amplification, or it may be added to a control reaction which is run in the same PCR apparatus under the same parameters. A control primer may comprise a sequence complementary to any identical sequence between a PKD gene and a PKD homologue. Preferably, the control primers generate a single amplified product whose size is distinguishable from that amplified by a pair of primers comprising at least one PKD-specific primers. The size of the amplified product by the control primers may be greater or smaller than the size of the amplified products generated by the pair of primers comprising at least one PKD-specific primers. Preferably, the control primers are chosen to generate a control product which has at least 100 bp, more preferably at least 500 bp, more preferably at least 1000 bp difference in size compared to the amplified product generated in the same amplification reaction by the pair of primers comprising at least one PKD-specific primers.

[0169] A control amplification is especially important when analyzing a PKD allele with deletions at the location where a PKD-specific primer anneals. The lack of a specific amplification in the presence of an amplified control product may indicate the presence of the deletion at a specific location of a PKD gene. In some embodiments, more than one pair of control primers is used in the reaction mixture.

[0170] See Example 2 for various controls that might be used for the genetic testing method of the invention.

[0171] Amplified products may be purified to get rid of free primers used in the amplification by methods known in the art (e.g., Current Protocols in Molecular Biology, supra). In a preferred embodiment, the PCR products are purified using the Quickstep™ 96 well PCR Purification Kit from Edge Biosystems.

[0172] VII. Detecting the Presence of PCR Amplified Products

[0173] The cycle of DNA denaturation, primer annealing and synthesis of the DNA segment defined by the 5′ ends of the primers is repeated as many times as is necessary to amplify the template target until a sufficient amount of either a species-specific or a universal product is available for detection. At the conclusion of the amplification reaction, the presence of amplified products may be detected using techniques conventional in the art.

[0174] The primers may be labeled for facilitating the detection. The primers can be labeled with a directly detectable tag, for example a radioactive label such as ³²P, ³⁵S, ¹⁴C or ¹²⁵I, a fluorescent compound such as fluorescein or rhodamine derivatives, an enzyme such as a peroxidase or alkaline phosphatase, or avidin or biotin. The PKD-specific primers used to generate the PKD-specific product and the control primers used only to generate the control product may have the same or different labels.

[0175] In a preferred embodiment, the amplification products are conveniently analyzed by gel electrophoresis.

[0176] Electrophoresis is conducted under conditions which effect a desired degree of resolution of fragments. A degree of resolution that separates fragments that differ in size by as little as about 500 bp is usually sufficient. Preferably, the resolution is at about 100 bp. More preferably, the resolution is at about 10 bp. Size markers may also be run on the gel to permit estimation of the size of fragments. Preliminary analysis of the size of specifically amplified products may indicate insertions or deletions within a PKD gene, and the information obtained can be interpreted together with results obtained from subsequent DHPLC and sequence analysis.

[0177] The amplification product pattern may be visualized. Where an amplification primer has been labeled, this label may be revealed. A substrate carrying the separated labeled DNA fragments is contacted with a reagent which detects the presence of the label. For example, an amplified product generated from a radioactively labeled primer may be detected by radioautography. Where the amplification primers are not labeled, the substrate bearing the PCR product may be contacted with ethidium bromide and the DNA fragments visualized under ultraviolet light.

[0178] VIII. Separating PCR Amplified Products

[0179] Under the most stringent condition which only allows the annealing of completely complementary sequences but not sequences comprising one or more non-complementary nucleotides, a PKD-specific primer will only anneal to an authentic PKD gene template, but not a PKD homologue. Therefore, under the most stringent condition, a PKD-specific primer, in combination with a primer with opposite orientation, being PKD-specific or not, will only produce amplified product from an authentic PKD template, but not from a PKD homologue. However, during a typical PCR amplification reaction, a PKD-specific may anneal to a template comprising an authentic PKD gene and a PKD homologue, especially due to the temperature cycling required by a PCR reaction. Therefore, both specifically amplified products and non-specifically amplified products may be produced, although the amount of non-specifically amplified products may be reduced by the use of at least one PKD-specific primer.

[0180] A. Formation of Homoduplex and Heteroduplex

[0181] In one embodiment of the invention, a mixture of homoduplexes and heteroduplexes is formed prior to the DHPLC analysis. A standard nucleic acid homoduplex (e.g., amplified product from a normal PKD allele) may be added to the sample and the mixture is subjected to denaturation, e.g. by heating the mixture to about 90° C. or about 95° C. The denatured single stranded nucleic acids formed during the denaturation process are then annealed by slowly cooling the mixture to ambient temperature. A new mixture of homoduplexes and heteroduplexes is formed if the sample contains a mutation. If the sample does not contain a mutation, only a homoduplex of the standard nucleic acid will be formed. In the preferred embodiment, the standard nucleic acid is the “normal” nucleic acid.

[0182] In most cases, a PKD patient individual is heterozygous at the loci comprising a PKD gene. That is, the carrier has only one PKD allele and a mutant form and has the other allele as a normal form (e.g., wild type). Since most of the PKD mutations result in a dominant phenotype, one mutant allele is sufficient to predispose a risk for ADPKD development. Another heterozygous situation is when both alleles are mutated but each carries one or more different mutations. For a heterozygous PKD patient, a PCR amplification using a primer pair comprising at least one PKD-specific primer, including a nested PCR amplification, would result in at least two specifically amplified PKD products, one from each allele. The two specifically amplified PKD products may or may not be of the same length (e.g., different length if the mutation on one allele comprises a deletion or an insertion) and would differ in at least one nucleotide from each other.

[0183] The amplified products may be denatured and re-annealed with each other to form duplexes. When a specifically amplified product from a normal allele or a specifically amplified product from a mutant allele anneals to another specifically amplified product from the same allele, they will form homoduplex. However, if a specifically amplified product from a normal allele anneals to a specifically amplified product from a mutant allele, they form a heteroduplex.

[0184] In rare cases, a mutation is in homozygous form, that is, both alleles in an individual (e.g., a PKD patient) comprise the same mutations. If a sample is taken from a homozygous PKD patient, the PCR amplification will not generate specifically amplified products which can form heteroduplex upon denaturing and re-annealing. In some embodiments of the invention, a sample comprising a normal (e.g., a wide type) PKD gene is added to the PCR reaction mixture so that amplification using a primer pair comprising at least one PKD-specific primer will produce specifically amplified products from the normal PKD gene, therefore ensuring the formation of a heteroduplex during the denaturation and re-annealing process following PCR amplification.

[0185] Homoduplexes formed in the denaturation and re-annealing process may also include those formed by non-specifically amplified products. If in very rare cases, a sequence in a template allele (e.g., a PKD homologue sequence) which give rise to non-specifically amplified products also comprises one or more mutation, a heteroduplex may also form. The heteroduplex formed between non-specifically amplified products will also be subjected to further separating the identification process.

[0186] B. Separating and Identifying Heteroduplex

[0187] The presence of a heteroduplex formed by PKD-specifically amplified products indicates the presence of a mutation in a PKD gene. By separating for heteroduplexes, one can identify whether a mutant allele present in the sample, e.g., taken from an individual. This separating process gets rid of most of the non-specifically amplified products and specifically amplified products from normal alleles, therefore improves the efficiency and specificity of identifying a mutant allele and a PKD patient.

[0188] It is well known in the DNA art that a heteroduplex strand will denature selectively at the site of base pair mismatch, creating a “bubble”, at a lower temperature than is necessary to denature the remainder of the heteroduplex strand, i.e., those portions of the heteroduplex strand which contain complimentary base pairs. This phenomenon, generally referred to as partial denaturation, occurs because the hydrogen bonds between mismatched bases are weaker than the hydrogen bonds between complimentary bases. Therefore, less energy is required to denature the heteroduplex at the mutation site, hence the lower temperature required to partially denature the heteroduplex at the site of base pair mismatch than in the remainder of the strand.

[0189] Since at least one base pair in a heteroduplex is not complimentary, it takes less energy to separate the bases at that site compared to its fully complimentary base pair analog in a homoduplex. This results in the lower melting temperature of a heteroduplex compared to a homoduplex. The local denaturation creates, what is generally called, a “bubble” at the site of base pair mismatch. The bubble distorts the structure of a DNA fragment compared to a fully complimentary homoduplex of the same base pair length. This structural distortion under partially denaturing conditions has serves as the basis for DHPLC to separate heteroduplexes and homoduplexes.

[0190] A separation process called “Denaturing HPLC” (DHPLC) has been used to detect mutations by separating a heteroduplex (resulting from the presence of a mutation) and a homoduplex having the same bp length. DHPLC has been applied to mutation detection (e.g., see Underhill, et al., 1997, Genome Research 7:996; Liu, et al., 1998, Nucleic Acid Res., 26;1396). This separation is based on the fact that a heteroduplex has a lower melting temperature (Tm) than a homoduplex. When DHPLC is carried out at a partially denaturing temperature, i.e., a temperature sufficient to denature a heteroduplex at the site of base pair mismatch, homoduplexes can be separated from heteroduplexes having the same base pair length (Hayward-Lester, et al., 1995, Genome Research, 5:494; Underhill, et al., 1996, Proc. Natl. Acad. Sci. USA 93:193; Doris, et al., 1997, DHPLC Workshop, Stanford University). These references and the references contained therein are incorporated herein in their entireties. Thus, the use of DHPLC was applied to mutation detection (Underhill, et al., 1997, Genome Research 7:996; Liu, et al., 1998, Nucleic Acid Res., 26:1396). DHPLC can separate heteroduplexes that differ by as little as one base pair under certain conditions. The references cited above and the references contained therein are incorporated in their entireties herein.

[0191] The change in the structure of DNA from an orderly helix to a disordered, unstacked structure without base pairs is called the helix-random chain transition, or melting. Statistical-mechanical analysis of equilibria representing this change as a function of temperature for double-stranded molecules of natural sequence has been presented by Wartell and Montroll (1972, Adv. Chem. Phys. 22: 129). The theory assumes that each base pair can exist in only two possible states-either stacked, helical, and hydrogen bonded, or disordered. It permits calculation of the probability that each individual base pair is either helical or melted at any temperature, given only the base sequence and a very small number of empirically calibrated parameters. The statistical-mechanical theories take into account the differing intrinsic stabilities of each base pair or cluster of neighboring base pairs, the influence of adjacent helical structure on the probability that a neighboring base pair is helical or melted (the coopertivity), and the restrictions on the conformational liberty of a disordered region if it is bounded at both ends by helical regions.

[0192] Iteration of the probability calculation at a closely spaced series of temperature steps and interpolation permit determination of the midpoint temperature at which each base pair is at 50/50 equilibrium between the helical and melted states. The MELT program provides the midpoint temperature and some other functions. A plot of midpoint temperature as a function of position along the molecule is called a melting map. It clearly shows that the melting of nearby base pairs is closely coupled over substantial lengths of the molecule despite their individual differences in stability. The existence of fairly long regions, 30-300 bp, termed domains, in which all bases melt at very nearly the same temperature, is typical. The melting map directly delineates the lowest melting domains in the molecules.

[0193] At a partially denaturing temperature, a heteroduplex having a base pair mismatch within a sample sequence will denature at the site of the mismatch, while the rest of the sample sequence will remain intact. The partially denatured heteroduplex can be separated and detected using DHPLC.

[0194] When HPLC is used under partially denaturing conditions (e.g., DHPLC) to separate a mixture of homoduplexes and heteroduplexes, the heteroduplexes usually elute ahead of the homoduplexes.

[0195] In particular embodiment of the invention, a heteroduplex is separated and identified from a homoduplex by DHPLC, and the presence of heteroduplex indicates the presence of at least one mutation in the PKD gene, e.g., a substitution of one or more nucleotides (or insertion or deletion of one or more nucleotides) present in the mutant allele.

[0196] In another particular embodiment, DHPLC gradient is determined by Wavemaker™ 4.0 software from Transgenomic, Inc. (San Jose, Calif.).

[0197] Separating applications require that the mutation can be detected regardless of where the mutation might be located on the fragment. In this situation, the mutation might be located in the middle of the fragment or in a higher melting domain, both cases where it is more difficult to detect. It is preferred than the range of melting variation of the fragment is no greater than 10° C. and most preferred is the range of variation is no greater than 5° C.

[0198] In some mutation analyses, only two peaks or a partially resolved peak(s) are observed in DHPLC analysis. The two homoduplex peaks may appear as one peak or a partially resolved peak and the two heteroduplex peaks may appear as one peak or a partially resolved peak. In some cases, only a broadening of the initial peak is observed under partially denaturing conditions.

[0199] If a sample contained homozygous DNA fragments of the same length, then hybridization and analysis by DHPLC would only produce a single peak at any temperature since no heteroduplexes could be formed. In the operation of the present method, the determination of a mutation can be made by hybridizing the homozygous sample with the known wild type fragment and performing a DHPLC analysis at a partially denaturing temperature. If the sample contained only normal allele then a single peak would be seen in the DHPLC analysis since no heteroduplexes could be formed. If the sample contained heterozygous mutant alleles, then analysis by DHPLC would show the separation of homoduplexes and heteroduplexes.

[0200] The temperature at which 50% of a constant melting domain is denatured may also be determined experimentally by plotting the UV (UV) absorbance of a DNA sample against temperature. The absorbance increases with temperature and the resulting plot is called a melting profile (Breslauer et al., 1986, Proc. Natl. Acad. Sci. USA 83:3746; Breslauer, 1987, Calculating Thermodynamic Data for Transitions of any Molecularity, p. 221, Marky et al. eds., J. Wiley and Sons). The midpoint of the absorbance axis on the melting profile represents the melting temperature (Tm), i.e. the temperature at which 50% of the DNA strands in the duplex are denatured. In one embodiment of the present invention, this observed Tm is used as a starting temperature for performing DHPLC for mutation detection. The temperature may be then adjusted according to the patterns observed using different controls (see below). In one embodiment, a consistent Tm is used to analyze the same amplicons (i.e., produced by the same pair of primers) from different samples.

[0201] In another embodiment of the present invention, software such as MELT (Lerman, et al., 1987, Meth. Enzymol. 155:482) or WinMelt™, version 2.0, is used to obtain a calculated Tm which is used as a starting temperature for performing DHPLC for mutation detection. These software programs show that despite individual differences in base pair stability, the melting temperature of nearby base pairs is closely coupled, i.e., there is a cooperative effect. Thus, there are long regions of 30 to 300 base pairs, called “domains”, in which the melting temperature is fairly constant. In a similar manner, the software MELTSCAN (Brossette, et al., 1994, Nucleic Acid Res. 22:4321) calculates melting domains in a DNA fragment and their corresponding melting temperatures. The concept of a constant temperature melting domain is important since it makes possible the detection of a mutation in any portion of the domain at a single heteromutant site selective temperature.

[0202] Another particular method for separating and identifying heteroduplex is Matched Ion Nucleic acid Chromatography (MIPC). MIPC was introduced to effectively separate mixtures of double stranded nucleic acids, in general and DNA, in particular, wherein the separations are based on base pair length (U.S. Pat. Nos. 5,585,236 and 6,287,822; Huber et al., 1993, Chromatographia 37:653; Huber et al., 1993, Anal. Biochem. 212:351). These references and the references contained therein are incorporated herein in their entireties. MIPC separations are complete in less than 10 minutes, and frequently in less than 5 minutes. MIPC systems (WAVE™ DNA Fragment Analysis System, Transgenomic, Inc. San Jose, Calif.) are equipped with computer controlled ovens which enclose the columns and column inlet areas.

[0203] Although DHPLC and MICP are the described methods for separating and identifying heteroduplex, it is understood that other methods known in the art may also be used for identifying heteroduplex. For example, heteroduplex analysis on high resolution gel matrices are also able to detect even single nucleotide polymorphisms. (Hauser et al., 1998, Plant. J. 16:117-25). The PCR/OLA procedure can be used for analyzing amplification products to detect SNPs in the 3′ end of the human PKD gene (Glick and Pasternak, 1994, Molecular Biotechnology: Principles and Applications of Recombinant DNA, ASM Press, Washington, D.C., pp. 197-200). Conformation-sensitive gel electrophoresis of amplification products may also be employed as a means of analysis by the skilled artisan in practicing the methods of the present invention. (Markoff et al., 1998, Eur. J. Genet. 6:145-50). This can also be achieved by techniques such as PCR-restriction fragment-SSCP, which can detect single base substitutions, deletions or insertions (Tawata et al., 1996, Genet. Anal. 12(3-4):125-27; Lee et al., 1992, Anal. Biochem. 205:289-93). Electrophoresis for analyzing amplification products is done rapidly and with high sensitivity by using any of various methods of conventional slab or capillary electrophoresis, with which the practitioner can optionally choose to employ any facilitating means of nucleic acid fragment detection, including, but not limited to, radionuclides, UV-absorbance or laser-induced fluorescence (Keparnik et al., 1998, Electrophoresis 19:249-55; Inoue et al. 1998, J. Chromatogr. A. 802:179-84; Dovichi, 1997, 18:2393-99; Arakawa et al., 1997, J. Pharm. Biomed. Anal. 15:1537-44; Baba, 1996, J. Chromatgr B. Biomed. Appl. 687:271-302; Chan et al., 1997, J. Chromatogr B. Biomed. Sci. Appl. 695:13-15). Any of diverse fluorescent dyes can optionally be used to label primers of the present invention or amplification products for ease of analysis, including but not limited to, SYBR Green I, Y1O-PRO-1, thiazole orange, Hex (i.e., 6-carboxy-2′,4′,7′,4,7-hexachlorofluoroscein), pico green, edans, fluorescein, FAM (i.e., 6-carboxyfluorescein), or TET (i.e., 4,7,2′,7′-tetrachloro-6-carboxyfluoroscein) (e.g., Skeidsvoll and Ueland, 1995, Anal. Biochem. 231:359-65; Iwahana et al., 1996, Biotechniques 21:510-14, 516-19).

[0204] In using the invention in its preferred embodiment to effect a separation of homoduplexes and heteroduplexes for the purpose of mutation detection, a DNA sample is hybridized with a normal DNA fragment by denaturing and annealing the mixture as described herein above. The DNA sample can be hybridized with normal DNA directly. The DNA sample can also be amplified by PCR and then hybridized with the normal DNA. Alternatively, a normal fragment may be added to the sample prior to PCR amplification. The amplified mixture can then be hybridized following amplification. In each of these three hybridization scenarios, a mixture of homoduplexes and heteroduplexes is produced if a mutation is present in the sample. The sample, so prepared, is analyzed by DHPLC under partially denaturing conditions, preferably at 56 to 58° C., for the presence of a mutation using the method of the invention.

[0205] When the method of the invention is used for separating a large number of samples for the presence of a mutation, the throughput of samples may be increased significantly by speeding up the analysis for each sample using a steeper gradient for the fragment bracketing range.

[0206] In all embodiments and aspects of the invention, the nucleic acid fragments are detected as they are separated and eluted from the DHPLC column. Any detector capable of detecting nucleic acids can be used in the DHPLC mutation detection method. The preferred detector is an online UV detector. If the DNA fragments are tagged with fluorescent or radioactive tags, then a fluorescence detector or radioactivity detector can be employed, respectively. Following detection, the separated fragments are displayed on a video display separate or printed by a printer. The fragments so displayed appear either as peaks or as bands in a lane.

[0207] C. Quality Controls Helpful for Evaluating DHPLC for PKD-2 and PKD-1 Unique Region

[0208] The chemical principles which permit DHPLC to distinguish between heteroduplex-homoduplex mixtures and homoduplexes alone also make the methodology quite sensitive to (1) buffer composition, (2) oven temperature at the time of analysis, (3) column condition, and (4) system condition at the time a sample is injected. Fluctuation in elution patterns is normal, and varies depending on the size and sequence of the amplicon, and the specific DHPLC conditions under which it is analyzed. On skilled in the art would have the knowledge in interpreting the elution patterns produced, for example, by following the protocol provided by the manufacture of the DHPLC equipment. However, limits on the extent of fluctuation are appropriate to help ensure that conditions are within a range that would be expected to effectively separate for DNA variants. The following quality control requirements are useful examples established for each analytical condition to ensure consistent assay performance.

[0209] 1. No DNA Control

[0210] This control demonstrates that reagents and materials are free of non-specific signal that could interfere with patient analysis. In some embodiment, the control must show minimal signal (<10% of normal control peak height) in a no-DNA sample treated identically to a sample comprising a DNA, e.g., extracted from a tissue. Because all of the analytical system's hardware is re-used for each sample analysis, and because the DHPLC analysis is the separating component, up to 10% peak height of the normal control is permitted. Actual contamination with a different sequence might cause a false positive DHPLC pattern difference which would trigger reflexing to sequencing which would not be expected to detect a 10% contaminant. In the event that a sequence difference is detected, the fragment would be repeated from the point of PCR to confirm the result. Similarly contamination of an actual positive with 10% of a normal sequence would not be expected to significantly alter the pattern since 50% of the DNA present is already normal. Rare cases where a very subtle pattern change might be obscured by 10% extra normal DNA in the injection are accounted for in the sensitivity estimates of 78-96%. However, persistent no DNA signal each time the amplicon is analyzed indicates the need to alter analytical conditions to minimize or eliminate a systematic and persistent no DNA signal.

[0211] 2. Normal Control

[0212] In one embodiment, the normal control pattern must be consistent with historic patterns. Consistency with established patterns indicates acceptable amplification, retention times, peak height, and peak shape. Therefore, PCR and DHPLC conditions (machine and buffers, etc.) are performed as specified in the Examples. Homologues, or other non-specific amplification signals are absent as indicated by comparison with the established normal control pattern.

[0213] 3. Positive Control

[0214] The positive controls are “DHPLC analytical condition controls” used to demonstrate that the established DHPLC analytical conditions (which detect the positive control heteroduplex) are in effect at the time of analysis. A positive control pattern distinct from normal control and consistent with historic patterns indicates acceptable retention time, peak height(s), peak shape and pattern. Heteroduplex detection indicates that the specific DHPLC analysis conditions optimal for the individual fragment were in effect during patient analysis. It is important to note that these controls are not necessarily PKD positive signals. Specific PKD positive samples for each of the 83 PKD fragments are not available. In their absence, another heteroduplex (positive and normal control) is used as the positive indicator demonstrating appropriate analytical conditions at the time of analysis.

[0215] 4. Additional Positive Controls

[0216] Additional positive controls provide pattern(s) consistent with historic patterns for this specific mutation and may be used to separate out very common polymorphisms. Generally, a specific DNA variant will generate a unique signature heteroduplex pattern that is highly reproducible from sample to sample. A pattern consistent with the established pattern indicates acceptable retention time, peak height(s), peak shape and pattern. The specific heteroduplex pattern demonstrates that specific DHPLC analysis conditions optimal for this DNA variant were in effect during patient analysis and, therefore, patient patterns matching this can be considered to possess the common polymorphism. This optional separating method for common polymorphisms is highly specific to the unique amplicon and variant and is dependent upon appropriate validation studies unique to the variant.

[0217] D. Analyzing DHPLC Results

[0218] Since DBPLC is a separating process, any specimen (e.g., DNA, or cell lysate or tissue sample) with a signal that differs from the normal control should be considered a potential positive and treated by one of several options available depending on the circumstances. For some embodiments, a signal that is too week to interpret (less than 25% of the normal control peak height) could be caused by PCR failure, Wave injection failure, or some other sporadic instrumentation problem unique to the sample. Options include repeat from the point of PCR, repeat the Wave injection (with all controls), or report the wave result as inconclusive and proceed to sequencing. A signal that differs from the normal control in pattern should be considered positive, scored as “P”, and sequenced. A signal that differs very slightly from the normal control pattern should be scored as “B” and sequenced. A signal that is much stronger than the normal control signal should be scored as “P” and sequenced. Note that no patient specimens will be resulted based on these results alone. The specific options utilized will vary with the amplicon and its DHPLC performance history, and the specific circumstances for the specimen.

[0219] In some embodiments, the only results released from the DHPLC results will be those scored as “normal” by Wave analysis. In order to be scored as normal, the specimen's DHPLC pattern must be consistent with the normal control by the following QC criteria: (a) peak number, (b) peak height, (c) peak pattern, (d) retention time, (e) baseline shape. In other words, the pattern for the individual specimen must look like the normal control, within a reasonable expected range of variation. Consult with the validation data reference patterns if necessary. The sensitivity of DHPLC separating was assessed by counting patterns that differ substantially from the normal control. When a pattern genuinely appears to differ from the normal control, there should be no doubt—it is scored as positive and sent on for sequencing. Only those that meet the requirements for that specific amplicon and have a pattern consistent with the normal control should be scored and released as normal.

[0220] Specific numerical criteria used for judging “consistent with” include, but are not limited to, (a) number of peaks where a peak represents a local maximum in the signal intensity, (b) peak heights, or maximum signal intensities, which are usually between 0.5 and 2.0 times the height of the normal control, (c) retention time of peaks, which must be +/−60 seconds compared to the corresponding normal controls. Peak pattern is judged by relative correspondence of each slope change within a peak, and relative intensities and retention times of individual peaks within a complex pattern. Baseline patterns are usually smooth and consistent in all samples. A relatively low baseline change may represent a heteroduplex that elutes and perhaps melts at considerably different retention times from the homoduplex peak(s). The retention time and peak height criteria for each amplicon are specified in the attached tables in the Examples.

[0221] In one embodiment, the peak pattern assessment is a combination of (1) the sample signal satisfying the same run control criteria as the normal control, and (2) the sample signal pattern being consistent with the normal control based on the relative comparison for that run. Normal control patterns are expected to vary slightly from run to run, and still be acceptable, so individual samples scored as normal are a combination of satisfying (1) the same run control criteria as the normal control, (2) the relative control criteria inherent in the comparison of the normal control to each patient sample, described above. It seems clear that subtle changes in the pattern of the patient sample might be consistent with the absolute run criteria for the normal control, yet be clearly distinct using relative comparison of normal and patient within a run. The relative comparison within a run always supercedes historic patterns, assuming the normal control has passed control criteria and the run is accepted.

[0222] IX. Verification of Heteroduplex

[0223] Optionally, the identified heteroduplex may be verified by means of digesting the amplification products with one or more restriction enzymes. The restriction enzymes useful for this purpose are selected by comparing the sequences of authentic PKD genes and PKD homologues, or by comparing PKD polymorphisms Useful restriction enzymes according to the invention generate distinguishable fragment profiles for an authentic PKD gene and a PKD homologue. Examples of such restriction enzymes include, but are not limited to, Pst I, Stu I, Xma I, Mlu I, Pvu II, BssHII, Fsp I, Msc I, and Bln I. Useful restriction enzymes may also generate distinguishable fragment profile for a normal PKD gene and a mutant PKD gene. It is understood that more restriction enzymes may be identified by simply comparing the sequence of a PKD gene and a PKD homologue gene or a normal PKD allele and a mutant PKD allele. A restriction enzyme with its recognition site or cleavage site in one sequence altered so as to abolish or create a cleavage site but not in the other sequence may be considered a useful restriction enzyme for the subject invention. Restriction of nucleic acids is followed by separation of the resulting fragments and analysis of fragment length or differential fragment migration in denaturing high-performance liquid chromatography (DHPLC) or gel electrophoresis, as above, including restriction-capillary electrophoresis.

[0224] X. Sequencing of Heteroduplexes Identified by DHPLC

[0225] Heteroduplex indicating the presence of one or more mutation, identified by DHPLC, may be cloned, amplified, and/or sequenced. Any known sequencing method known in the art can be used to sequence the heteroduplex. In some embodiments, the heteroduplex identified was used as template for PCR amplification and amplified products are sequenced by Sequetech Corporation (Mountain View, Calif.). In a preferred embodiment, sequencing is carried out by using one of the primers with SEQ ID NOs. 3-49.

[0226] In some embodiments, the identified heteroduplex is amplified and cloned into a plasmid (e.g., Zero Blunt TOPO PCR cloning kit, Invitrogen, Carlsbad, Calif., Cat #4560-01) before sequencing. The plasmid containing the PCR fragment is then propagated by well known methods in the art before subject to sequencing.

[0227] XI. Clinical Use of the Method

[0228] The genetic testing method described in this application is targeted toward identifying DNA alterations in the coding region of the PKD-1 or PKD-2 gene, including the splice junction acceptor/donor sequences, which have been reported to cause ADPKD. The method can be performed to assists physicians to:

[0229] A. Diagnose PKD-caused ADPKD in symptomatic individuals.

[0230] B. Follow up on ultrasound results indicating the presence of one or two cysts in an individual at or near the age of onset.

[0231] C. Diagnose between different variants of ADPKD (type 1 and 2), which is not feasible to determine from family history, ultrasound and other clinical data.

[0232] D. Determine and provide genetic counseling for other at-risk family members once an ADPKD proband has been identified in a family.

[0233] E. Determine the suitability of a living related donor in transplantation cases.

[0234] XII. Kits

[0235] The invention also provides kits for performing the mutation analysis method and the PKD patient identification method of the invention. Embodiments of the subject kits, in accordance with the methods of the invention, include at least one isolated first nucleic acid and at least one isolated second nucleic acid, where the first nucleic acid is selected from the group of SEQ ID NOs. 3-49 and their complementary sequences, and the second nucleic acid has an opposite orientation from the first nucleic acid, and where the first and second nucleic acids amplify a fragment of a template nucleic acid comprising a sequence of SEQ ID NO. 1 or 2, and packaging materials therefore. The kit of the invention may further comprises at least one component selected from the group consisting of: a DNA polymerase, a template nucleic acid, a restriction enzyme, a control oligonucleotide primer, ddNTPs, a PCR reaction buffer and the combination thereof. Kits of the invention, in addition to the reagents, preferably include written instructions for performing the subject methods. Kits are preferably packaged in a unit container and may contain the reagents in pre-measured amounts designed to operate with each other so as to produce the desired result.

EXAMPLES

[0236] The invention is illustrated by the following non-limiting examples wherein the following materials and methods are employed.

Example 1

[0237] Reagents, Special Supplies and Equipment

[0238] A. Chemicals

[0239] The following is a listed of chemicals used for PKD-1 amplification and DHPLC (WAVE) analysis.

[0240] 1% Agarose, 1×TBE, 54 Well Gel with Ethidium Bromide (Embitec, Catalog Number GE 4580)

[0241] 2% Agarose, 1×TBE, 54 Well Gel with Ethidium Bromide (Embitec, Catalog Number GE 4582)

[0242] 96 Well Gel Filtration Block (Edge Biosystems, Catalog Number 91751)

[0243] Quickstep™ 96 Well PCR Purification Kit (Edge Biosystems, Catalog Number 99605)

[0244] AmpliTaq Gold with GeneAmp PCR Buffer II & MgCl₂ Solution (Perkin Elmer, Catalog Number N808-0241)

[0245] rTth DNA Polymerase, XL & XL Buffer II Pack (Perkin Elmer, Catalog Number N808-00193)

[0246] TapPlus Precision PCR System (Stratagene, Catalog Number 600211)

[0247] Dimethyl Sulphoxide (DMSO) (Sigma, Catalog Number D-2650)

[0248] Ready-Load 100 bp DNA Ladder or Equivalent (Gibco BRL, Catlaog Number 10380-012)

[0249] Ready-Load 1 kb DNA Ladder or Equivalent (Gibco BRL, 1800-828-6686, Catlaog Number 10381-010)

[0250] Big Dye Terminator Ready Reaction Kit (Perkin Elmer, Catalog Number 4303150)

[0251] Gel Filtration Cartridge (Edge Biosystems, Catalog Number 42453)

[0252] Long Ranger Singel™ packs (FMC BioProducts, Catalog Number 50691 or 50693).

[0253] Oligonucleotides (Operon Technologies, Inc.)

[0254] WAVE Mutation Standard (209 bp), Catalog Number 560077 (180 ul)

[0255] Acetonitrile-HPLC Grade (VWR, Catalog Number BJ015-1)

[0256] HPLC Grade Water (VWR, Catalog Number BJ365-4)

[0257] Triethylammoniun Acetate (TEAA) (Transgenomic, Catalog Number SP5890)

[0258] B. Reagents and Solutions

[0259] 10 μM oligonucleotide primers: 10 μM working aliquots of PCR primers dissolved in TE buffer should be stored at 4° C. in Pre-PCR refrigerator; sequencing primer working aliquots should be stored at 4° C. in Post-PCR refrigerator.

[0260] Solution X-127: Upgrade Blue Dextran in 50 mM EDTA (pH=8.0)

[0261] Combine 0.5 ml 50 mM EDTA pH=8.0 (Solution X-35), 500 mg Blue Dextran AND 9.5 ml AUTOCLAVED, STERILE FILTERED DiH₂O in a sterile 15 ml conical centrifuge tube. Thoroughly mix the solution by vortexing.

[0262] Solution X-126: Upgrade Gel Loading Buffer: Combine 200 μl deionized Formamide and 40 μl Upgrade Blue Dextran in 50 mM EDTA (Solution X-127) in a 1.5 ml sterile microcentrifuge tube. Vortex thoroughly.

[0263] WAVE Solution A: Solution A (0.025% ACN) Preparation of 2L: 100 ml Ion Pairing Agent (TEAA) 500 μl Acetonitrile (ACN) Top to 2L with HPLC grade water

[0264] WAVE Solution B: Solution B (25% ACN) Preparation of 2L: 100 ml Ion Pairing Agent (TEAA) 500 ml Acetonitrile (ACN) Top to 2L with HPLC grade water

[0265] WAVE Syringe Wash Solution: _Syringe Wash (8% ACN) Preparation of 2L: 160 ml Acetonitrile (ACN) Top to 2L with HPLC grade water

[0266] WAVE Solution D: Solution D (75% ACN) Preparation of 2L: 500 ml HPLC grade water Top to 2L with Acetonitrile (ACN)

[0267] C. Equipment and Special Supplies TABLE 5 Perkin Elmer ABI Prism ™ 377 DNA Sequencer 761 Main Avenue Norwalk, CT 06859 VWR Scientific Products 1. Beckman Allegra ™ 21 Centrifuge P.O. Box 232 2. Eppendorf Microcentrifuge 5415C Boston, MA 02101 3. Multichannel pipet 4. Sterile reservoirs 5. DURX 670 wipers 6. VWR Model 1300U Oven Transgenomic, Inc. WAVE Nucleic Acid Fragment 12325 Emmet Street Analysis System Omaha, NE 68164

Example 2

[0268] Procedure

[0269] Stage I: Preparation of DNA and/or RNA from Patient Specimens

[0270] DNA is extracted from whole blood or lymphocytes using the Puregene® DNA extraction kit. DNA extracted using these reagents should be successfully PCR amplified under the conditions specific to the assay. This is tested by performing the assay as specified in the protocol and comparing the results obtained with the positive DNA control that has been previously validated.

[0271] Extracted DNA is quantitated and the 260/280 ratio is 1.4 or greater. Samples with lower ratios indicate that the quality of DNA is poor and may not meet PCR standards. If end results of the assay are not interpretable the sample should be re-extracted.

[0272] Stage II: Amplification of DNA by PCR

[0273] PCR reaction mixtures and cycling parameters (e.g., for exon 1 of PKD-1 gene) were set up as illustrated in Table 5. PCR conditions were set up similarly, but optimized for specific and efficient amplification of other exons. TABLE 6 PCR Reaction Master Mix Component Concentrations and Thermal Cycling Conditions For First round PCR Products 1-8 (L1-L8) LOWER MASTER MIX: Reaction Volume/ Component: Concentration reaction Water — 13.0 ul 10X Buffer 1X  2.0 ul Mg(Oac)₂  0.9 mM None dNTP mix  200 uM  1.0 ul Primer 1 0.25 uM 1.25 ul Primer 2 0.25 uM 1.25 u1 DMSO 7.5%  1.5 ul TOTAL   20 ul VOLUME

[0274] One wax bead was added to each well and incubated in a thermal cycler @ 80° C. for 5 minutes to melt the wax and incubated at 25° C. for an additional 5 minutes before placed on ice for further handling. UPPER MASTER MIX: Reaction Volume/ Component: Concentration reaction Water — 23.15 ul 10X Buffer 1X  3.0 ul TaqPlus Precision Polymerase mixture 5U/rxn  1.0 ul DMSO 7.5%  2.25 ul TOTAL VOLUME  29.4 ul Genomic  0.6 ul DNA @ 500 ng/ul

[0275] Cycling Parameters Melting the Wax Amplification 80° C. 5 min 1 cycle 94° C.  3 min  1 cycle 25° C. forever 96° C. 30 sec 68° C. 20 sec 35 cycles 72° C.  3 min + 4 sec/cycle 72° C. 10 min  1 cycle

[0276] TABLE 7 Example of nested PCR reaction setup STOCK REACTION CONCEN- VOLUME PER CONCEN- REAGENT TRATION REACTION TRATION Water — 31.0 μl — Buffer II 10X  5.0 μl 1X MgCl₂ 25 mM  2.0 μl  1.0 mM DNTP mix 10 mM each  1.0 μl  200 μM each CAD-18-PF1 (primer) 10 μM  3.0 μl  0.6 μM CAD-18-PR1 10 μM  3.0 μl  0.6 μM (primer) DMSO 100%  2.5 μl 5% Amplitaq Gold  5 U/μl  0.5 μl  2.5 U TOTAL 48.0 μl

[0277] TABLE 8 Summary of Amplification Conditions For Exons CYCLE NUMBER TEMPERATURE TIME DESCRIPTION  1 cycle 94° C. 10 min AmpliTaq Gold activation 92° C.  1 min Denaturing 35 cycles 55° C.  1 min Annealing 72° C.  1 min Extension  1 cycle 72° C. 10 min Final extension (hold)  4° C. forever

[0278] PCR amplified fragments may be compared in size, signal intensity and migration pattern with positive control DNA control that has been previously validated. The size of the PCR amplified fragments is determined by comparison to the Molecular weight marker (DNA MASS™ Ladder-Gibco BRL) on the gel. The low range DNA Mass Ladder gives 6 bands of double stranded (100-2000 bp) DNA on staining the gel with ethidium bromide.

[0279] Stage III: DHPLC Analysis of PCR Products

[0280] Heteroduplexes formed by PCR amplified products are analyzed using WAVE nucleic acid fragment analysis system from Transgenomic, Inc. (Omaha, NE 68164).

[0281] Stage IV: Cycle Sequencing

[0282] Tables 9 and 10 provide examples of sequencing conditions used in one embodiment of the invention. TABLE 9 Sequencing Reaction Master Mix Component STOCK REACTION CONCEN- VOLUME PER CONCEN- REAGENT TRATION REACTION TRATION Water — 14.0 μl — Big Dye Terminator 2.5X  4.0 μl 0.5X Ready Reaction Mix Primer 10 μM  1.0 μl 0.5 μM FINAL VOLUME 19.0 μl

[0283] TABLE 10 Cycle Sequencing Conditions CYCLE NUMBER TEMPERATURE TIME DESCRIPTION 94° C. 10 sec Denaturing 30 cycles 55° C.  5 sec Annealing 60° C.  4 min Extension (hold)  4° C. forever

Example 3

[0284] Summary of Results

[0285] In one experiment, detection of mutations in exons 1-34 of the PKD-1 gene was achieved by using eight sets of oligonucleotide primers in eight separate first round PCR reaction to amplify DNA fragments of the following sizes: a) LR1 was 2.2 kb and contains exon 1. b) LR2 was 4.6 kb and contains exons 2-7. c) LR3 was 4.2 kb and contains exons 8-12. d) LR4 was 4.4 kb and contains exons 13-15. e) LR5 was 3.4 kb and contains exons 15 (3′-end) through 21. f) LR6 was 0.3 kb and consists of exon 22. g) LR7 was 4.2 kb and contains exons 23-28. h) LR8 was 5.8 kb and contained exons 29-34 of the duplicated region of the gene. The amplified product from the first round of amplification were then serially diluted to 1:10⁴ or 1:10⁵ to remove genomic contamination and subsequently used as template in a second round of nested PCR. The nested PCR products were heteroduplexed and screened for sequence alterations by DHPLC. Each fragment was analyzed against a normal and positive control using a temperature and acetonitrile gradient specific to the amplicon. Any samples testing positive by DHPLC analysis were subsequently purified and sequenced. Cycle sequenced products were then separated on an ABI 377 automated sequencer and the results were analyzed using an assortment of sequencing software. Tables 11-12 and FIGS. 1 to 13 illustrate the results and procedures of some embodiments of the invention. TABLE 11 Numbers of products analyzed for each PKD gene Analysis: PKD-1 PKD-2 Total First Round PCRs 8 — 8 Amplicons 66 17 83 DHPLC analyses 133 33 166 Base Pairs evaluated 13,830 3204 17,034

[0286] TABLE 12 Variant detection rates Naturally occuring - Naturally Independent occuring - Mutagenesis Source of Sequence SSCP Sequence Variant confirmed Separated confirmed Gene Total PKD-1 14/18 15/17 45/47 74/82 78% 88% 96% 90% PKD-2 20/21 0/0 22/23 42/44 95% 96% 95% Type total 34/39 15/17 67/70 116/126 87% 88% 96% 92%

Other Embodiments

[0287] The foregoing examples demonstrate experiments performed and contemplated by the present inventors in making and carrying out the invention. It is believed that these examples include a disclosure of techniques which serve to both apprise the art of the practice of the invention and to demonstrate its usefulness. It will be appreciated by those of skill in the art that the techniques and embodiments disclosed herein are preferred embodiments only and that in general numerous equivalent methods and techniques may be employed to achieve the same result. All applications, patents and literature referred to in the specification are hereby incorporated by reference, in their entirety, including figures and tables.

1 168 1 14136 DNA Homo sapiens 1 gcactgcagc gccagcgtcc gagcgggcgg ccgagctccc ggagcggcct ggccccgagc 60 cccgagcggg cgtcgctcag cagcaggtcg cggccgcgca gccccatcca gccccgcgcc 120 cgccatgccg tccgcgggcc ccgcctgagc tgcggtctcc gcgcgcgggc gggcctgggg 180 acggcggggc catgcgcgcg ctgccctaac gatgccgccc gccgcgcccg cccgcctggc 240 gctggccctg ggcctgggcc tgtggctcgg ggcgctggcg gggggccccg ggcgcggctg 300 cgggccctgc gagcccccct gcctctgcgg cccagcgccc ggcgccgcct gccgcgtcaa 360 ctgctcgggc cgcgggctgc ggacgctcgg tcccgcgctg cgcatccccg cggacgccac 420 agcgctagac gtctcccaca acctgctccg ggcgctggac gttgggctcc tggcgaacct 480 ctcggcgctg gcagagctgg atataagcaa caacaagatt tctacgttag aagaaggaat 540 atttgctaat ttatttaatt taagtgaaat aaacctgagt gggaacccgt ttgagtgtga 600 ctgtggcctg gcgtggctgc cgcgatgggc ggaggagcag caggtgcggg tggtgcagcc 660 cgaggcagcc acgtgtgctg ggcctggctc cctggctggc cagcctctgc ttggcatccc 720 cttgctggac agtggctgtg gtgaggagta tgtcgcctgc ctccctgaca acagctcagg 780 caccgtggca gcagtgtcct tttcagctgc ccacgaaggc ctgcttcagc cagaggcctg 840 cagcgccttc tgcttctcca ccggccaggg cctcgcagcc ctctcggagc agggctggtg 900 cctgtgtggg gcggcccagc cctccagtgc ctcctttgcc tgcctgtccc tctgctccgg 960 ccccccgcca cctcctgccc ccacctgtag gggccccacc ctcctccagc acgtcttccc 1020 tgcctcccca ggggccaccc tggtggggcc ccacggacct ctggcctctg gccagctagc 1080 agccttccac atcgctgccc cgctccctgt cactgccaca cgctgggact tcggagacgg 1140 ctccgccgag gtggatgccg ctgggccggc tgcctcgcat cgctatgtgc tgcctgggcg 1200 ctatcacgtg acggccgtgc tggccctggg ggccggctca gccctgctgg ggacagacgt 1260 gcaggtggaa gcggcacctg ccgccctgga gctcgtgtgc ccgtcctcgg tgcagagtga 1320 cgagagcctt gacctcagca tccagaaccg cggtggttca ggcctggagg ccgcctacag 1380 catcgtggcc ctgggcgagg agccggcccg agcggtgcac ccgctctgcc cctcggacac 1440 ggagatcttc cctggcaacg ggcactgcta ccgcctggtg gtggagaagg cggcctggct 1500 gcaggcgcag gagcagtgtc aggcctgggc cggggccgcc ctggcaatgg tggacagtcc 1560 cgccgtgcag cgcttcctgg tctcccgggt caccaggagc ctagacgtgt ggatcggctt 1620 ctcgactgtg cagggggtgg aggtgggccc agcgccgcag ggcgaggcct tcagcctgga 1680 gagctgccag aactggctgc ccggggagcc acacccagcc acagccgagc actgcgtccg 1740 gctcgggccc accgggtggt gtaacaccga cctgtgctca gcgccgcaca gctacgtctg 1800 cgagctgcag cccggaggcc cagtgcagga tgccgagaac ctcctcgtgg gagcgcccag 1860 tggggacctg cagggacccc tgacgcctct ggcacagcag gacggcctct cagccccgca 1920 cgagcccgtg gaggtcatgg tattcccggg cctgcgtctg agccgtgaag ccttcctcac 1980 cacggccgaa tttgggaccc aggagctccg gcggcccgcc cagctgcggc tgcaggtgta 2040 ccggctcctc agcacagcag ggaccccgga gaacggcagc gagcctgaga gcaggtcccc 2100 ggacaacagg acccagctgg cccccgcgtg catgccaggg ggacgctggt gccctggagc 2160 caacatctgc ttgccgctgg acgcctcttg ccacccccag gcctgcgcca atggctgcac 2220 gtcagggcca gggctacccg gggcccccta tgcgctatgg agagagttcc tcttctccgt 2280 tgccgcgggg ccccccgcgc agtactcggt caccctccac ggccaggatg tcctcatgct 2340 ccctggtgac ctcgttggct tgcagcacga cgctggccct ggcgccctcc tgcactgctc 2400 gccggctccc ggccaccctg gtccccaggc cccgtacctc tccgccaacg cctcgtcatg 2460 gctgccccac ttgccagccc agctggaggg cacttgggcc tgccctgcct gtgccctgcg 2520 gctgcttgca gccacggaac agctcaccgt gctgctgggc ttgaggccca accctggact 2580 gcggatgcct gggcgctatg aggtccgggc agaggtgggc aatggcgtgt ccaggcacaa 2640 cctctcctgc agctttgacg tggtctcccc agtggctggg ctgcgggtca tctaccctgc 2700 cccccgcgac ggccgcctct acgtgcccac caacggctca gccttggtgc tccaggtgga 2760 ctctggtgcc aacgccacgg ccacggctcg ctggcctggg ggcagtgtca gcgcccgctt 2820 tgagaatgtc tgccctgccc tggtggccac cttcgtgccc ggctgcccct gggagaccaa 2880 cgataccctg ttctcagtgg tagcactgcc gtggctcagt gagggggagc acgtggtgga 2940 cgtggtggtg gaaaacagcg ccagccgggc caacctcagc ctgcgggtga cggcggagga 3000 gcccatctgt ggcctccgcg ccacgcccag ccccgaggcc cgtgtactgc agggagtcct 3060 agtgaggtac agccccgtgg tggaggccgg ctcggacatg gtcttccggt ggaccatcaa 3120 cgacaagcag tccctgacct tccagaacgt ggtcttcaat gtcatttatc agagcgcggc 3180 ggtcttcaag ctctcactga cggcctccaa ccacgtgagc aacgtcaccg tgaactacaa 3240 cgtaaccgtg gagcggatga acaggatgca gggtctgcag gtctccacag tgccggccgt 3300 gctgtccccc aatgccacgc tagcactgac ggcgggcgtg ctggtggact cggccgtgga 3360 ggtggccttc ctgtggaact ttggggatgg ggagcaggcc ctccaccagt tccagcctcc 3420 gtacaacgag tccttcccgg ttccagaccc ctcggtggcc caggtgctgg tggagcacaa 3480 tgtcatgcac acctacgctg ccccaggtga gtacctcctg accgtgctgg catctaatgc 3540 cttcgagaac ctgacgcagc aggtgcctgt gagcgtgcgc gcctccctgc cctccgtggc 3600 tgtgggtgtg agtgacggcg tcctggtggc cggccggccc gtcaccttct acccgcaccc 3660 gctgccctcg cctgggggtg ttctttacac gtgggacttc ggggacggct cccctgtcct 3720 gacccagagc cagccggctg ccaaccacac ctatgcctcg aggggcacct accacgtgcg 3780 cctggaggtc aacaacacgg tgagcggtgc ggcggcccag gcggatgtgc gcgtctttga 3840 ggagctccgc ggactcagcg tggacatgag cctggccgtg gagcagggcg cccccgtggt 3900 ggtcagcgcc gcggtgcaga cgggcgacaa catcacgtgg accttcgaca tgggggacgg 3960 caccgtgctg tcgggcccgg aggcaacagt ggagcatgtg tacctgcggg cacagaactg 4020 cacagtgacc gtgggtgcgg ccagccccgc cggccacctg gcccggagcc tgcacgtgct 4080 ggtcttcgtc ctggaggtgc tgcgcgttga acccgccgcc tgcatcccca cgcagcctga 4140 cgcgcggctc acggcctacg tcaccgggaa cccggcccac tacctcttcg actggacctt 4200 cggggatggc tcctccaaca cgaccgtgcg ggggtgcccg acggtgacac acaacttcac 4260 gcggagcggc acgttccccc tggcgctggt gctgtccagc cgcgtgaaca gggcgcatta 4320 cttcaccagc atctgcgtgg agccagaggt gggcaacgtc accctgcagc cagagaggca 4380 gtttgtgcag ctcggggacg aggcctggct ggtggcatgt gcctggcccc cgttccccta 4440 ccgctacacc tgggactttg gcaccgagga agccgccccc acccgtgcca ggggccctga 4500 ggtgacgttc atctaccgag acccaggctc ctatcttgtg acagtcaccg cgtccaacaa 4560 catctctgct gccaatgact cagccctggt ggaggtgcag gagcccgtgc tggtcaccag 4620 catcaaggtc aatggctccc ttgggctgga gctgcagcag ccgtacctgt tctctgctgt 4680 gggccgtggg cgccccgcca gctacctgtg ggatctgggg gacggtgggt ggctcgaggg 4740 tccggaggtc acccacgctt acaacagcac aggtgacttc accgttaggg tggccggctg 4800 gaatgaggtg agccgcagcg aggcctggct caatgtgacg gtgaagcggc gcgtgcgggg 4860 gctcgtcgtc aatgcaagcc gcacggtggt gcccctgaat gggagcgtga gcttcagcac 4920 gtcgctggag gccggcagtg atgtgcgcta ttcctgggtg ctctgtgacc gctgcacgcc 4980 catccctggg ggtcctacca tctcttacac cttccgctcc gtgggcacct tcaatatcat 5040 cgtcacggct gagaacgagg tgggctccgc ccaggacagc atcttcgtct atgtcctgca 5100 gctcatagag gggctgcagg tggtgggcgg tggccgctac ttccccacca accacacggt 5160 acagctgcag gccgtggtta gggatggcac caacgtctcc tacagctgga ctgcctggag 5220 ggacaggggc ccggccctgg ccggcagcgg caaaggcttc tcgctcaccg tgctcgaggc 5280 cggcacctac catgtgcagc tgcgggccac caacatgctg ggcagcgcct gggccgactg 5340 caccatggac ttcgtggagc ctgtggggtg gctgatggtg accgcctccc cgaacccagc 5400 tgccgtcaac acaagcgtca ccctcagtgc cgagctggct ggtggcagtg gtgtcgtata 5460 cacttggtcc ttggaggagg ggctgagctg ggagacctcc gagccattta ccacccatag 5520 cttccccaca cccggcctgc acttggtcac catgacggca gggaacccgc tgggctcagc 5580 caacgccacc gtggaagtgg atgtgcaggt gcctgtgagt ggcctcagca tcagggccag 5640 cgagcccgga ggcagcttcg tggcggccgg gtcctctgtg cccttttggg ggcagctggc 5700 cacgggcacc aatgtgagct ggtgctgggc tgtgcccggc ggcagcagca agcgtggccc 5760 tcatgtcacc atggtcttcc cggatgctgg caccttctcc atccggctca atgcctccaa 5820 cgcagtcagc tgggtctcag ccacgtacaa cctcacggcg gaggagccca tcgtgggcct 5880 ggtgctgtgg gccagcagca aggtggtggc gcccgggcag ctggtccatt ttcagatcct 5940 gctggctgcc ggctcagctg tcaccttccg cctgcaggtc ggcggggcca accccgaggt 6000 gctccccggg ccccgtttct cccacagctt cccccgcgtc ggagaccacg tggtgagcgt 6060 gcggggcaaa aaccacgtga gctgggccca ggcgcaggtg cgcatcgtgg tgctggaggc 6120 cgtgagtggg ctgcagatgc ccaactgctg cgagcctggc atcgccacgg gcactgagag 6180 gaacttcaca gcccgcgtgc agcgcggctc tcgggtcgcc tacgcctggt acttctcgct 6240 gcagaaggtc cagggcgact cgctggtcat cctgtcgggc cgcgacgtca cctacacgcc 6300 cgtggccgcg gggctgttgg agatccaggt gcgcgccttc aacgccctgg gcagtgagaa 6360 ccgcacgctg gtgctggagg ttcaggacgc cgtccagtat gtggccctgc agagcggccc 6420 ctgcttcacc aaccgctcgg cgcagtttga ggccgccacc agccccagcc cccggcgtgt 6480 ggcctaccac tgggactttg gggatgggtc gccagggcag gacacagatg agcccagggc 6540 cgagcactcc tacctgaggc ctggggacta ccgcgtgcag gtgaacgcct ccaacctggt 6600 gagcttcttc gtggcgcagg ccacggtgac cgtccaggtg ctggcctgcc gggagccgga 6660 ggtggacgtg gtcctgcccc tgcaggtgct gatgcggcga tcacagcgca actacttgga 6720 ggcccacgtt gacctgcgcg actgcgtcac ctaccagact gagtaccgct gggaggtgta 6780 tcgcaccgcc agctgccagc ggccggggcg cccagcgcgt gtggccctgc ccggcgtgga 6840 cgtgagccgg cctcggctgg tgctgccgcg gctggcgctg cctgtggggc actactgctt 6900 tgtgtttgtc gtgtcatttg gggacacgcc actgacacag agcatccagg ccaatgtgac 6960 ggtggccccc gagcgcctgg tgcccatcat tgagggtggc tcataccgcg tgtggtcaga 7020 cacacgggac ctggtgctgg atgggagcga gtcctacgac cccaacctgg aggacggcga 7080 ccagacgccg ctcagtttcc actgggcctg tgtggcttcg acacagaggg aggctggcgg 7140 gtgtgcgctg aactttgggc cccgcgggag cagcacggtc accattccac gggagcggct 7200 ggcggctggc gtggagtaca ccttcagcct gaccgtgtgg aaggccggcc gcaaggagga 7260 ggccaccaac cagacggtgc tgatccggag tggccgggtg cccattgtgt ccttggagtg 7320 tgtgtcctgc aaggcacagg ccgtgtacga agtgagccgc agctcctacg tgtacttgga 7380 gggccgctgc ctcaattgca gcagcggctc caagcgaggg cggtgggctg cacgtacgtt 7440 cagcaacaag acgctggtgc tggatgagac caccacatcc acgggcagtg caggcatgcg 7500 actggtgctg cggcggggcg tgctgcggga cggcgaggga tacaccttca cgctcacggt 7560 gctgggccgc tctggcgagg aggagggctg cgcctccatc cgcctgtccc ccaaccgccc 7620 gccgctgggg ggctcttgcc gcctcttccc actgggcgct gtgcacgccc tcaccaccaa 7680 ggtgcacttc gaatgcacgg gctggcatga cgcggaggat gctggcgccc cgctggtgta 7740 cgccctgctg ctgcggcgct gtcgccaggg ccactgcgag gagttctgtg tctacaaggg 7800 cagcctctcc agctacggag ccgtgctgcc cccgggtttc aggccacact tcgaggtggg 7860 cctggccgtg gtggtgcagg accagctggg agccgctgtg gtcgccctca acaggtcttt 7920 ggccatcacc ctcccagagc ccaacggcag cgcaacgggg ctcacagtct ggctgcacgg 7980 gctcaccgct agtgtgctcc cagggctgct gcggcaggcc gatccccagc acgtcatcga 8040 gtactcgttg gccctggtca ccgtgctgaa cgagtacgag cgggccctgg acgtggcggc 8100 agagcccaag cacgagcggc agcaccgagc ccagatacgc aagaacatca cggagactct 8160 ggtgtccctg agggtccaca ctgtggatga catccagcag atcgctgctg cgctggccca 8220 gtgcatgggg cccagcaggg agctcgtatg ccgctcgtgc ctgaagcaga cgctgcacaa 8280 gctggaggcc atgatgctca tcctgcaggc agagaccacc gcgggcaccg tgacgcccac 8340 cgccatcgga gacagcatcc tcaacatcac aggagacctc atccacctgg ccagctcgga 8400 cgtgcgggca ccacagccct cagagctggg agccgagtca ccatctcgga tggtggcgtc 8460 ccaggcctac aacctgacct ctgccctcat gcgcatcctc atgcgctccc gcgtgctcaa 8520 cgaggagccc ctgacgctgg cgggcgagga gatcgtggcc cagggcaagc gctcggaccc 8580 gcggagcctg ctgtgctatg gcggcgcccc agggcctggc tgccacttct ccatccccga 8640 ggctttcagc ggggccctgg ccaacctcag tgacgtggtg cagctcatct ttctggtgga 8700 ctccaatccc tttccctttg gctatatcag caactacacc gtctccacca aggtggcctc 8760 gatggcattc cagacacagg ccggcgccca gatccccatc gagcggctgg cctcagagcg 8820 cgccatcacc gtgaaggtgc ccaacaactc ggactgggct gcccggggcc accgcagctc 8880 cgccaactcc gccaactccg ttgtggtcca gccccaggcc tccgtcggtg ctgtggtcac 8940 cctggacagc agcaaccctg cggccgggct gcatctgcag ctcaactata cgctgctgga 9000 cggccactac ctgtctgagg aacctgagcc ctacctggca gtctacctac actcggagcc 9060 ccggcccaat gagcacaact gctcggctag caggaggatc cgcccagagt cactccaggg 9120 tgctgaccac cggccctaca ccttcttcat ttccccgggg agcagagacc cagcggggag 9180 ttaccatctg aacctctcca gccacttccg ctggtcggcg ctgcaggtgt ccgtgggcct 9240 gtacacgtcc ctgtgccagt acttcagcga ggaggacatg gtgtggcgga cagaggggct 9300 gctgcccctg gaggagacct cgccccgcca ggccgtctgc ctcacccgcc acctcaccgc 9360 cttcggcgcc agcctcttcg tgcccccaag ccatgtccgc tttgtgtttc ctgagccgac 9420 agcggatgta aactacatcg tcatgctgac atgtgctgtg tgcctggtga cctacatggt 9480 catggccgcc atcctgcaca agctggacca gttggatgcc agccggggcc gcgccatccc 9540 tttctgtggg cagcggggcc gcttcaagta cgagatcctc gtcaagacag gctggggccg 9600 gggctcaggt accacggccc acgtgggcat catgctgtat ggggtggaca gccggagcgg 9660 ccaccggcac ctggacggcg acagagcctt ccaccgcaac agcctggaca tcttccggat 9720 cgccaccccg cacagcctgg gtagcgtgtg gaagatccga gtgtggcacg acaacaaagg 9780 gctcagccct gcctggttcc tgcagcacgt catcgtcagg gacctgcaga cggcacgcag 9840 cgccttcttc ctggtcaatg actggctttc ggtggagacg gaggccaacg ggggcctggt 9900 ggagaaggag gtgctggccg cgagcgacgc agcccttttg cgcttccggc gcctgctggt 9960 ggctgagctg cagcgtggct tctttgacaa gcacatctgg ctctccatat gggaccggcc 10020 gcctcgtagc cgtttcactc gcatccagag ggccacctgc tgcgttctcc tcatctgcct 10080 cttcctgggc gccaacgccg tgtggtacgg ggctgttggc gactctgcct acagcacggg 10140 gcatgtgtcc aggctgagcc cgctgagcgt cgacacagtc gctgttggcc tggtgtccag 10200 cgtggttgtc tatcccgtct acctggccat cctttttctc ttccggatgt cccggagcaa 10260 ggtggctggg agcccgagcc ccacacctgc cgggcagcag gtgctggaca tcgacagctg 10320 cctggactcg tccgtgctgg acagctcctt cctcacgttc tcaggcctcc acgctgaggc 10380 ctttgttgga cagatgaaga gtgacttgtt tctggatgat tctaagagtc tggtgtgctg 10440 gccctccggc gagggaacgc tcagttggcc ggacctgctc agtgacccgt ccattgtggg 10500 tagcaatctg cggcagctgg cacggggcca ggcgggccat gggctgggcc cagaggagga 10560 cggcttctcc ctggccagcc cctactcgcc tgccaaatcc ttctcagcat cagatgaaga 10620 cctgatccag caggtccttg ccgagggggt cagcagccca gcccctaccc aagacaccca 10680 catggaaacg gacctgctca gcagcctgtc cagcactcct ggggagaaga cagagacgct 10740 ggcgctgcag aggctggggg agctggggcc acccagccca ggcctgaact gggaacagcc 10800 ccaggcagcg aggctgtcca ggacaggact ggtggagggt ctgcggaagc gcctgctgcc 10860 ggcctggtgt gcctccctgg cccacgggct cagcctgctc ctggtggctg tggctgtggc 10920 tgtctcaggg tgggtgggtg cgagcttccc cccgggcgtg agtgttgcgt ggctcctgtc 10980 cagcagcgcc agcttcctgg cctcattcct cggctgggag ccactgaagg tcttgctgga 11040 agccctgtac ttctcactgg tggccaagcg gctgcacccg gatgaagatg acaccctggt 11100 agagagcccg gctgtgacgc ctgtgagcgc acgtgtgccc cgcgtacggc caccccacgg 11160 ctttgcactc ttcctggcca aggaagaagc ccgcaaggtc aagaggctac atggcatgct 11220 gcggagcctc ctggtgtaca tgctttttct gctggtgacc ctgctggcca gctatgggga 11280 tgcctcatgc catgggcacg cctaccgtct gcaaagcgcc atcaagcagg agctgcacag 11340 ccgggccttc ctggccatca cgcggtctga ggagctctgg ccatggatgg cccacgtgct 11400 gctgccctac gtccacggga accagtccag cccagagctg gggcccccac ggctgcggca 11460 ggtgcggctg caggaagcac tctacccaga ccctcccggc cccagggtcc acacgtgctc 11520 ggccgcagga ggcttcagca ccagcgatta cgacgttggc tgggagagtc ctcacaatgg 11580 ctcggggacg tgggcctatt cagcgccgga tctgctgggg gcatggtcct ggggctcctg 11640 tgccgtgtat gacagcgggg gctacgtgca ggagctgggc ctgagcctgg aggagagccg 11700 cgaccggctg cgcttcctgc agctgcacaa ctggctggac aacaggagcc gcgctgtgtt 11760 cctggagctc acgcgctaca gcccggccgt ggggctgcac gccgccgtca cgctgcgcct 11820 cgagttcccg gcggccggcc gcgccctggc cgccctcagc gtccgcccct ttgcgctgcg 11880 ccgcctcagc gcgggcctct cgctgcctct gctcacctcg gtgtgcctgc tgctgttcgc 11940 cgtgcacttc gccgtggccg aggcccgtac ttggcacagg gaagggcgct ggcgcgtgct 12000 gcggctcgga gcctgggcgc ggtggctgct ggtggcgctg acggcggcca cggcactggt 12060 acgcctcgcc cagctgggtg ccgctgaccg ccagtggacc cgtttcgtgc gcggccgccc 12120 gcgccgcttc actagcttcg accaggtggc gcagctgagc tccgcagccc gtggcctggc 12180 ggcctcgctg ctcttcctgc ttttggtcaa ggctgcccag cagctacgct tcgtgcgcca 12240 gtggtccgtc tttggcaaga cattatgccg agctctgcca gagctcctgg gggtcacctt 12300 gggcctggtg gtgctcgggg tagcctacgc ccagctggcc atcctgctcg tgtcttcctg 12360 tgtggactcc ctctggagcg tggcccaggc cctgttggtg ctgtgccctg ggactgggct 12420 ctctaccctg tgtcctgccg agtcctggca cctgtcaccc ctgctgtgtg tggggctctg 12480 ggcactgcgg ctgtggggcg ccctacggct gggggctgtt attctccgct ggcgctacca 12540 cgccttgcgt ggagagctgt accggccggc ctgggagccc caggactacg agatggtgga 12600 gttgttcctg cgcaggctgc gcctctggat gggcctcagc aaggtcaagg agttccgcca 12660 caaagtccgc tttgaaggga tggagccgct gccctctcgc tcctccaggg gctccaaggt 12720 atccccggat gtgcccccac ccagcgctgg ctccgatgcc tcgcacccct ccacctcctc 12780 cagccagctg gatgggctga gcgtgagcct gggccggctg gggacaaggt gtgagcctga 12840 gccctcccgc ctccaagccg tgttcgaggc cctgctcacc cagtttgacc gactcaacca 12900 ggccacagag gacgtctacc agctggagca gcagctgcac agcctgcaag gccgcaggag 12960 cagccgggcg cccgccggat cttcccgtgg cccatccccg ggcctgcggc cagcactgcc 13020 cagccgcctt gcccgggcca gtcggggtgt ggacctggcc actggcccca gcaggacacc 13080 ccttcgggcc aagaacaagg tccaccccag cagcacttag tcctccttcc tggcgggggt 13140 gggccgtgga gtcggagtgg acaccgctca gtattacttt ctgccgctgt caaggccgag 13200 ggccaggcag aatggctgca cgtaggttcc ccagagagca ggcaggggca tctgtctgtc 13260 tgtgggcttc agcactttaa agaggctgtg tggccaacca ggacccaggg tcccctcccc 13320 agctcccttg ggaaggacac agcagtattg gacggtttct agcctctgag atgctaattt 13380 atttccccga gtcctcaggt acagcgggct gtgcccggcc ccaccccctg ggcagatgtc 13440 ccccactgct aaggctgctg gcttcaggga gggttagcct gcaccgccgc caccctgccc 13500 ctaagttatt acctctccag ttcctaccgt actccctgca ccgtctcact gtgtgtctcg 13560 tgtcagtaat ttatatggtg ttaaaatgtg tatatttttg tatgtcacta ttttcactag 13620 ggctgagggg cctgcgccca gagctggcct cccccaacac ctgctgcgct tggtaggtgt 13680 ggtggcgtta tggcagcccg gctgctgctt ggatgcgagc ttggccttgg gccggtgctg 13740 ggggcacagc tgtctgccag gcactctcat caccccagag gccttgtcat cctcccttgc 13800 cccaggccag gtagcaagag agcagcgccc aggcctgctg gcatcaggtc tgggcaagta 13860 gcaggactag gcatgtcaga ggaccccagg gtggttagag gaaaagactc ctcctggggg 13920 ctggctccca gggtggagga aggtgactgt gtgtgtgtgt gtgtgcgcgc gcgacgcgcg 13980 agtgtgctgt atggcccagg cagcctcaag gccctcggag ctggctgtgc ctgcttctgt 14040 gtaccacttc tgtgggcatg gccgcttcta gagcctcgac acccccccaa cccccgcacc 14100 aagcagacaa agtcaataaa agagctgtct gactgc 14136 2 6749 DNA Homo sapiens misc_feature (1)..(6749) “n” at position 719, 1277, 1278, 1279, 1280, 1288, 1289, 1638, 1967, 2248, 2251, 2254, 2283, 2585, 2586, 2625, 2932, 2949, 2972, 2978, 3406, is any of A, T, G, and C. 2 ggctcctgag gcgcacagcg ccgagcgcgg cgccgcgcac ccgcgcgccg gacgccagtg 60 accgcgatgg tgaactccag tcgcgtgcag cctcagcagc ccggggacgc caagcggccg 120 cccgcgcccc gcgcgccgga cccgggccgg ctgatggctg gctgcgcggc cgtgggcgcc 180 agcctcgccg ccccgggccg cctctgcgag cagcggggcc tggagatcga gatgcagcgc 240 atccggcagg cggccgcgcg ggaccccccg gccggagccg cggcctcccc ttctcctccg 300 ctctcgtcgt gctcccggca ggcgtggagc cgcgataacc ccggcttcga ggccgaggag 360 gaggaggagg aggtggaagg ggaagaaggc ggaatggtgg tggagatgga cgtagagtgg 420 cgcccgggca gccggaggtc ggccgcctcc tcggccgtga gctccgtggg cgcgcggagc 480 cgggggcttg ggggctacca cggcgcgggc cacccgagcg ggaggcggcg ccggcgagag 540 gaccagggcc cgccgtgccc cagcccagtc ggcggcgggg acccgctgca tcgccacctc 600 cccctggaag ggcagccgcc ccgagtggcc tgggcggaga ggctggttcg cgggctgcga 660 ggtgtaagag cgcgcgaccc gcagcggcag atgcacgaac cagaacggcc ggcgccggng 720 gcttcttaaa taaaatgata tcttttcttt tcttcattat tattttaaag gtctctgggg 780 aacaagactc atggaggaaa gcagcactaa ccgagagaaa taccttaaaa gtgttttacg 840 ggaactggtc acatacctcc tttttctcat agtcttgtgc atctgtaagt agaatatttc 900 cttgcactaa tgggaaagtt ttgaaacgat gtgaatttgt ccaaaatgtt tatccacagg 960 aacaatccct ttgtgaaggc tgctggtatg tggatgtgtg ccggttccct tggggcgttc 1020 atttggatct ttctgtgttc cagtgaccta cggcatgatg agctccaatg tgtactacta 1080 cacccggatg atgtcacagc tcttcctaga cacccccgtg tccaaaacgg agaaaactaa 1140 ctttaaaact ctgtcttcca tggaagactt ctggaaggta tttggaaata actttgaaag 1200 tacctctcta tcacaagcca atgcttggtt atgcaacgat gcaggcaggg caaagcagcg 1260 gcatgagctt gaacttnnnn agatgttnnc tttcttttag ttcacagaag gctccttatt 1320 ggatgggctg tactggaaga tgcagcccag caaccagact gaagctgaca accgaagttt 1380 catcttctat gagaacctgc tgttaggggt tccacgaata cggcaactcc gagtcagaaa 1440 tggatcctgc tctatccccc aggacttgag agatgaaatt aaagagtgct atgatgtcta 1500 ctctgtcagt agtgaagata gggctccctt tgggccccga aatggaaccg cgtaagtgtc 1560 tgtgactcat tggcactcgg tgatattcat ccttgtaatt gcctcaagtg ttccactgat 1620 tgtaactgtt tgtttttngg ttttgttttt aatcagttgg atctacacaa gtgaaaaaga 1680 cttgaatggt agtagccact ggggaatcat tgcaacttat agtggagctg gctattatct 1740 ggatttgtca agaacaagag aggaaacagc tgcacaagtt gctagcctca agaaaaatgt 1800 ctggctggac cgaggaacca gggcaacttt tattgacttc tcagtgtaca acgccaacat 1860 taacctgttc tgtgtggtca ggtgtgtgac tgaggacatg catccctcct atttctgtgt 1920 ggttgtacat acatcctatt ctagggttac ccagaaaaac ctttttntgc aggttgttat 1980 tgttttaatt gttcttattt acatgcaggt tattggttga attcccagca acaggtggtg 2040 tgattccatc ttggcaattt cagcctttaa agctgatccg atatgtcaca acttttgatt 2100 tcttcctggc agcctgtgag attatctttt gtttctttat cttttactat gtggtggaag 2160 agatattgga aattcgcatt cacaaactac actatttcag gagtttctgg aattgtctgg 2220 atgttgtgat cgttgtggta ggtccganca ncancaccaa atttcctatt ctattctaca 2280 agnatgttaa caattaatac attggtgaag aaaaatatac tagtcatatt aaggtaagtt 2340 tcatatttct aaaacactgt aataaaatat aaatattttg cttttcagct gtcagtggta 2400 gctataggaa ttaacatata cagaacatca aatgtggagg tgctactaca gtttctggaa 2460 gatcaaaata ctttccccaa ctttgagcat ctggcatatt ggcagataca gttcaacaat 2520 atagctgctg tcacagtatt ttttgtctgg attaaggtaa tttataaatt tcatgttcta 2580 cattnnaaat aatattttct ttaaaaaaaa tgagttccac aaaancatgc gaaacaatgt 2640 tttattatac acagtcacac catttggttt atccattcat ctattgatgt cttctctctc 2700 ttacagctct tcaaattcat caattttaac aggaccatga gccagctctc gacaaccatg 2760 tctcgatgtg ccaaagacct gtttggcttt gctattatgt tcttcattat tttcctagcg 2820 tatgctcagt tggcatacct tgtctttggc actcaggtcg atgacttcag tactttccaa 2880 gagtgtatgt aagtatatat gaaattaaga agaaaaattt agtcagagta gncactgttg 2940 cgtggacant ctttggtttt gtattgtggt gntttgtntt atttttatag cttcactcaa 3000 ttccgtatca ttttgggcga tatcaacttt gcagagattg aggaagctaa tcgagttttg 3060 ggaccaattt atttcactac atttgtgttc tttatgttct tcattctttt ggtatgtaca 3120 tttatattta tagtggaggt tcaatttaaa cttcgtaaat ccttgtcttc tcttttttga 3180 ttgataattc caaattatgt ttcttccttt aatttttgcc ctcctttcat ttacaaacag 3240 aatatgtttt tggctatcat caatgatact tactctgaag tgaaatctga cttggcacag 3300 cagaaagctg aaatggaact ctcagatctt atcagaaagg taggaaaaac cttaattctc 3360 aaaaattctt ctgtttctga cataaaatga gcattgtttc acccanattt tagaatacnc 3420 taaaccaagt cttttatttt ttctctctct gatagggcta ccataaagct ttggtcaaac 3480 taaaactgaa aaaaaatacc gtggatgaca tttcagagag tctgcggcaa ggaggaggca 3540 agttaaactt tgacgaactt cgacaagatc tcaaagggtg agaatcatgc ttcctgaggt 3600 tctnaaaaat tcctgcttct aaagataaat tcctggtgat aagagtattt ctagcccaag 3660 ggctcatggg aacanaggat gaatgttatc tgtatcctct ctctaatttc aggaagggcc 3720 atactgatgc agagattgag gcaatattca caaagtacga ccaagatgga gaccaagaac 3780 tgaccgaaca tgaacatcag cagatgagag acgacttgga gaaagagagg gtgggtctgg 3840 tttaggagna accggatttg atttggtacc tacaacacca cacttctgtg gggtctcagt 3900 gttctgctcc tcactcagtg accccttgtt cttcaggagg acctggattt ggatcacagt 3960 tctttaccac gtcccatgag cagccgaagt ttccctcgaa gcctggatga ctctgaggag 4020 gatgacgatg aagatagcgg acatagctcc agaaggaggg gaagcatttc tagtggcgtt 4080 tcttacgaag agtttcaagt gtaagtataa aggaattggc agaatttgcg tngacaattt 4140 gtccctctgt actgtgtttt ccttgcagcc tggtgagacg agtggaccgg atggagcatt 4200 ccatcggcag catagtgtcc aagattgacg ccgtgatcgt gaagctagag attatggagc 4260 gagccaaact gaagaggagg gaggtgctgg gaaggctgtt ggatggggtg gccgaggtca 4320 gtagtcatga gctgaanaca ccgctgctga gcatggtgtt attaatnnna atatatgttg 4380 ctgacagttg tatttnaagt attnactgac ccccaacacc agtttctttt tcccttttta 4440 ggatgaaagg ctgggtcgtg acagtgaaat ccatagggaa cagatggaac ggctagtacg 4500 tgaagagttg gaacgctggg aatccgatga tgcagcttcc cagatcagtc atggtttagg 4560 cacgccagtg ggactaaatg gtcaacctcg ccccagaagc tcccgcccat cttcctccca 4620 atctacagaa ggcatggaag gtgcaggtgg aaatgggagt tctaatgtcc acgtatgata 4680 tgtgtgtttc agtatgtgtg tttctaataa gtgaggaagt ggctgtcctg aattgctgta 4740 acaagcacac tatttatatg ccctgaccac cataggatgc tagtctttgt gaccgattgc 4800 taatcttctg cactttaatt tattttatat aaactttacc catggttcaa agattttttt 4860 ttctttttct catataagaa atctaggtgt aaatattgag tacagaaaaa aaatcttcat 4920 gatgtgtatt gagcggtacg cccagttgcc accatgactg agtcttctca gttgacaatg 4980 aagtagcctt ttaaagctag aaaactgtca aagggcttct gagtttcatt tccagtcaca 5040 aaaatcagta ttgttatttt tttccaagag tgtgaaggaa aatggggcaa ttcctttcca 5100 ctctggcata gttcatgagc ttaatacata gctttctttt aagaaaggag cctttttttt 5160 caactagctt cctggggtaa acttttctaa aagataaaat gggaaggaac tccaaactat 5220 gatagaatct gtgtgaatgg ttaagatgaa tgttaaatac tatgcttttt tgtaagttga 5280 tcgtatctga tgtctgtggg actaactgta tcacttaatt tttaccttat tttggctcta 5340 atttgaataa gctgagtaaa accaccaaag atcagttata ggataaaatg gcatctctaa 5400 ccataacaca ggagaattgg aaggagccct aagttgtcac tcagtttaat ttcttttaat 5460 ggttagttta gcctaaagat ttatctgcat attctttttc ccatgtggct ctactcattt 5520 gcaactgaat ttaatgttat aactcatcta gtgagaccaa cttactaaat ttttagtatg 5580 cactgaaagt ttttatccaa caattatgtt cattttaagc aaaattttaa gaaagttttg 5640 aaattcataa agcatttggt tttaaactat tttaagaata tagtactcgg tcaggtatgn 5700 nncacgcctg taatcccagc actttgggag gccgaaacag gcgaatcact tgagcccagg 5760 agttcaagac caacatgggc aatgtggcga aactccatct ctacaaaaaa tgcaaaaata 5820 aaaaatatag tactcaagta ttcttgatcc tgtgtttcaa aactagaatt tgtaatgcaa 5880 atggagctca gtctaataaa aaagaggttt tggtattaaa agttcataca ttagacagta 5940 tcagccaaaa tttgagttag caacactgtt ttctttacga gagggtctca cccaaattta 6000 tggggagaaa tctatttctc aaaaaaaaaa aatcttcttt tacagaaatg ttgagtaagg 6060 tgacattttg agcgctaata agcaaaagag catgcagtgc tgttgaataa ccctcacttg 6120 gagaaccaag agaatcctgt cgtttaatgc tatattttaa tttcacaagt tgttcattta 6180 actggtagaa tgtcagtcca atctccaatg agaacatgag caaatagacc tttccaggtt 6240 gaaagtgaaa catactgggt ttctgtaagt ttttcctcat ggcttcatct ctatctttac 6300 tttctcttga atatgctaca caaagttctt tattactaca tactaaagtt tgcattccag 6360 ggatattgac tgtacatatt tatgtatatg taccatgttg ttacatgtaa acaaacttca 6420 atttgaagtg cagctattat gtggtatcca tgtgtatcga ccatgtgcca tatatcaatt 6480 atggtcacta gaaagtctct ttatgatact ttttattgta ctgtttttca tttcacttgc 6540 aaaattttgc agaattcctc ctttctaccc ataaattaca tataattttt cttctttagt 6600 catggagaac ncccccccat catctcancc ctattanctt tcccatgtgt actggtatta 6660 ttaaaaagac atttacatac gcaagttttt cactgacaan caagaatgtt attaatgtgt 6720 aatactgagc acntttactt cttaataaa 6749 3 20 DNA Artificial Sequence misc_feature (1)..(20) Synthetic primer 3 tggctgcaac tgcctcctgg 20 4 23 DNA Artificial Sequence misc_feature (1)..(23) Synthetic primer 4 aagcagagac agacctgtga gag 23 5 34 DNA Artificial Sequence misc_feature (1)..(34) Synthetic primer 5 gcccccgccg ctctcacagg tctgtctctg cttc 34 6 20 DNA Artificial Sequence misc_feature (1)..(20) Synthetic primer 6 ggcctgtagc ctacccctgg 20 7 19 DNA Artificial Sequence misc_feature (1)..(19) Synthetic primer 7 ggacccctct gaagccacc 19 8 21 DNA Artificial Sequence misc_feature (1)..(21) Synthetic primer 8 gggaggtggg agacaagaga c 21 9 21 DNA Artificial Sequence misc_feature (1)..(21) Synthetic primer 9 aaagccctgc tgtcactgtg g 21 10 23 DNA Artificial Sequence misc_feature (1)..(23) Synthetic primer 10 aactaaagcc cagaagacag acc 23 11 21 DNA Artificial Sequence misc_feature (1)..(21) Synthetic primer 11 aactgtctgc cccagaacat c 21 12 23 DNA Artificial Sequence misc_feature (1)..(23) Synthetic primer 12 ctaaaggctg ctctctcaac aag 23 13 22 DNA Artificial Sequence misc_feature (1)..(22) Synthetic primer 13 actcctgttg ggttttgatg ag 22 14 23 DNA Artificial Sequence misc_feature (1)..(23) Synthetic primer 14 gagaactact cccttgtcct tgg 23 15 24 DNA Artificial Sequence misc_feature (1)..(24) Synthetic primer 15 acgccaagga caagggagta gttc 24 16 22 DNA Artificial Sequence misc_feature (1)..(22) Synthetic primer 16 tgggctcctg gctggtgact gc 22 17 40 DNA Artificial Sequence misc_feature (1)..(40) Synthetic primer 17 gcggcccgcc gcccccgccg ctactgaccc gcaccctctg 40 18 18 DNA Artificial Sequence misc_feature (1)..(18) Synthetic primer 18 gctgcgaggg gtgagacg 18 19 42 DNA Artificial Sequence misc_feature (1)..(42) Synthetic primer 19 gcggcccgcc gcccccgccg cgtccctccc gccctcctga cc 42 20 36 DNA Artificial Sequence misc_feature (1)..(36) Synthetic primer 20 gcccccgccg ctgcggacga gaaatctgtc tgcttg 36 21 20 DNA Artificial Sequence misc_feature (1)..(20) Synthetic primer 21 cagggctgca agcagacaga 20 22 20 DNA Artificial Sequence misc_feature (1)..(20) Synthetic primer 22 ctgagctaag acgccctccc 20 23 21 DNA Artificial Sequence misc_feature (1)..(21) Synthetic primer 23 ctgtacgccc tcactggtgt c 21 24 21 DNA Artificial Sequence misc_feature (1)..(21) Synthetic primer 24 ggcacagggg ctcagtcagt c 21 25 21 DNA Artificial Sequence misc_feature (1)..(21) Synthetic primer 25 ggactgactg agcccctgtg c 21 26 20 DNA Artificial Sequence misc_feature (1)..(20) Synthetic primer 26 agtcggtcaa actgggtgag 20 27 20 DNA Artificial Sequence misc_feature (1)..(20) Synthetic primer 27 caaggtgtga gcctgagccc 20 28 21 DNA Artificial Sequence misc_feature (1)..(21) Synthetic primer 28 cggtgtccac tccgactcca c 21 29 32 DNA Artificial Sequence misc_feature (1)..(32) Synthetic primer 29 ccgcccccgc cgcgcgccgg acgccagtga cc 32 30 30 DNA Artificial Sequence misc_feature (1)..(32) Synthetic primer 30 gcccccgccg ccgcggcctc cccttctcct 30 31 44 DNA Artificial Sequence misc_feature (1)..(44) Synthetic primer 31 cggcccgccg cccccgcccg cggccgttct ggttcgtgca tctg 44 32 34 DNA Artificial Sequence misc_feature (1)..(34) Synthetic primer 32 gcccccgccg aaatgatatc ttttcttttc ttca 34 33 30 DNA Artificial Sequence misc_feature (1)..(30) Synthetic primer 33 cccccgcccg aactttccca ttagtgcaag 30 34 36 DNA Artificial Sequence misc_feature (1)..(36) Synthetic primer 34 cgccgccccc gcccgtgtga tagagaggta ctttca 36 35 39 DNA Artificial Sequence misc_feature (1)..(39) Synthetic primer 35 ccgccgcccc cgccgctttt tcaaagatgt ttcctttgc 39 36 20 DNA Artificial Sequence misc_feature (1)..(20) Synthetic primer 36 tatcaccgag tgccaatgag 20 37 35 DNA Artificial Sequence misc_feature (1)..(35) Synthetic primer 37 ccgccgcccc cgccggcctc aagtgttcca ctgat 35 38 35 DNA Artificial Sequence misc_feature (1)..(35) Synthetic primer 38 cccccgcccg ttgtagaata gaataggaaa tttgg 35 39 35 DNA Artificial Sequence misc_feature (1)..(35) Synthetic primer 39 gcccccgccg ttggtgaaga aaaatatact agtca 35 40 37 DNA Artificial Sequence misc_feature (1)..(37) Synthetic primer 40 cgccgccccc gcccgtggaa ctcatttttt ttaaaga 37 41 39 DNA Artificial Sequence misc_feature (1)..(39) Synthetic primer 41 gcgggggcgg cgggccgttt tattatacac agtcacacc 39 42 32 DNA Artificial Sequence misc_feature (1)..(32) Synthetic primer 42 gcccccgccg cttcctttaa tttttgccct cc 32 43 39 DNA Artificial Sequence misc_feature (1)..(39) Synthetic primer 43 cgccgccccc gcccggaaac aatgctcatt ttatgtcag 39 44 39 DNA Artificial Sequence misc_feature (1)..(39) Synthetic primer 44 ccgccgcccc cgccgaaacc aagtctttta ttttttctc 39 45 39 DNA Artificial Sequence misc_feature (1)..(39) Synthetic primer 45 ccgccgcccc cgccggatga atgttatctg tatcctctc 39 46 37 DNA Artificial Sequence misc_feature (1)..(37) Synthetic primer 46 cgccgccccc gcccggcaaa ttctgccaat tccttta 37 47 32 DNA Artificial Sequence misc_feature (1)..(32) Synthetic primer 47 gcccccgccg tttgtccctc tgtactgtgt tt 32 48 32 DNA Artificial Sequence misc_feature (1)..(32) Synthetic primer 48 ccgcccccgc cgtgaccccc aacaccagtt tc 32 49 40 DNA Artificial Sequence misc_feature (1)..(40) Synthetic primer 49 cggcccgccg cccccgcccg ggacagccac ttcctcactt 40 50 20 DNA Artificial Sequence misc_feature (1)..(20) Synthetic primer 50 cgtcgctcag cagcaggtcg 20 51 19 DNA Artificial Sequence misc_feature (1)..(19) Synthetic primer 51 cgtcctgctt cccgtcccg 19 52 42 DNA Artificial Sequence misc_feature (1)..(42) Synthetic primer 52 gcggcccgcc gcccccgccg ttggggatgc tggcaatgtg tg 42 53 20 DNA Artificial Sequence misc_feature (1)..(20) Synthetic primer 53 gggattcggc aaagctgatg 20 54 20 DNA Artificial Sequence misc_feature (1)..(20) Synthetic primer 54 ttccatcagc tttgccgaat 20 55 21 DNA Artificial Sequence misc_feature (1)..(21) Synthetic primer 55 atctggtctc aagcctggaa g 21 56 49 DNA Artificial Sequence misc_feature (1)..(49) Synthetic primer 56 gccccgcgcc cgtcccgccg cccccgccga gacccttccc accagacct 49 57 31 DNA Artificial Sequence misc_feature (1)..(31) Synthetic primer 57 cgcccccgcc cgtgagccct gcccagtgtc t 31 58 41 DNA Artificial Sequence misc_feature (1)..(41) Synthetic primer 58 gcggcccgcc gcccccgccg gagccaggag gagcagaacc c 41 59 22 DNA Artificial Sequence misc_feature (1)..(22) Synthetic primer 59 cagagggaca ggcaggcaaa gg 22 60 28 DNA Artificial Sequence misc_feature (1)..(28) Synthetic primer 60 gcccccgccg cccagccctc cagtgcct 28 61 20 DNA Artificial Sequence misc_feature (1)..(20) Synthetic primer 61 atcgctatgt gctgcctggg 20 62 18 DNA Artificial Sequence misc_feature (1)..(18) Synthetic primer 62 ccgaggtgga tgccgctg 18 63 21 DNA Artificial Sequence misc_feature (1)..(21) Synthetic primer 63 gaaggggagt gggcagcaga c 21 64 21 DNA Artificial Sequence misc_feature (1)..(21) Synthetic primer 64 cactgaccgt tgacaccctc g 21 65 21 DNA Artificial Sequence misc_feature (1)..(21) Synthetic primer 65 tgccccagtg cttcagagat c 21 66 19 DNA Artificial Sequence misc_feature (1)..(19) Synthetic primer 66 ggagtgccct gagccccct 19 67 19 DNA Artificial Sequence misc_feature (1)..(19) Synthetic primer 67 cccctaacca cagccagcg 19 68 21 DNA Artificial Sequence misc_feature (1)..(21) Synthetic primer 68 tctgttcgtc ctggtgtcct g 21 69 21 DNA Artificial Sequence misc_feature (1)..(21) Synthetic primer 69 gcaggagggc aggttgtaga a 21 70 40 DNA Artificial Sequence misc_feature (1)..(40) Synthetic primer 70 gcggcccgcc gcccccgccg ggtaggggga gtctgggctt 40 71 17 DNA Artificial Sequence misc_feature (1)..(17) Synthetic primer 71 gaggccaccc cgagtcc 17 72 20 DNA Artificial Sequence misc_feature (1)..(20) Synthetic primer 72 gttgggcatc tctgacggtg 20 73 35 DNA Artificial Sequence misc_feature (1)..(35) Synthetic primer 73 cgccgccccc gcccgggaag gtggcctgag gagat 35 74 37 DNA Artificial Sequence misc_feature (1)..(37) Synthetic primer 74 gcggcccgcc gcccccgccg ggggtccacg ggccatg 37 75 20 DNA Artificial Sequence misc_feature (1)..(20) Synthetic primer 75 aagcccagca gcacggtgag 20 76 34 DNA Artificial Sequence misc_feature (1)..(34) Synthetic primer 76 ccgccgcccc cgccgctgcc ctgcctgtgc cctg 34 77 51 DNA Artificial Sequence misc_feature (1)..(51) Synthetic primer 77 gccccgcgcc cgtcccgccg cccccgcccg ttccaccacc acgtccacca c 51 78 21 DNA Artificial Sequence misc_feature (1)..(21) Synthetic primer 78 gtggtggacg tggtggtgga a 21 79 21 DNA Artificial Sequence misc_feature (1)..(21) Synthetic primer 79 ggctgctgcc ctcactggga a 21 80 21 DNA Artificial Sequence misc_feature (1)..(21) Synthetic primer 80 taagggcaga gtcctccaca g 21 81 22 DNA Artificial Sequence misc_feature (1)..(22) Synthetic primer 81 ccacccccgc ccacctactg ag 22 82 40 DNA Artificial Sequence misc_feature (1)..(40) Synthetic primer 82 gcggcccgcc gcccccgccg tggagggagg gacgccaatc 40 83 19 DNA Artificial Sequence misc_feature (1)..(19) Synthetic primer 83 gaggctgggg ctgggacaa 19 84 18 DNA Artificial Sequence misc_feature (1)..(18) Synthetic primer 84 cccggttcac tcactgcg 18 85 30 DNA Artificial Sequence misc_feature (1)..(30) Synthetic primer 85 cccccgcccg ccgtgctcag agcctgaaag 30 86 38 DNA Artificial Sequence misc_feature (1)..(38) Synthetic primer 86 ggcggggggc ttctgccgag cgggtgggga gcaggtgg 38 87 36 DNA Artificial Sequence misc_feature (1)..(36) Synthetic primer 87 cgccgccccc gcccggctct gggtcaggac agggga 36 88 18 DNA Artificial Sequence misc_feature (1)..(18) Synthetic primer 88 cgcctggggg tgttcttt 18 89 18 DNA Artificial Sequence misc_feature (1)..(18) Synthetic primer 89 acgtgatgtt gtcgcccg 18 90 32 DNA Artificial Sequence misc_feature (1)..(32) Synthetic primer 90 gcccccgccg gggcgccccc gtggtggtca gc 32 91 18 DNA Artificial Sequence misc_feature (1)..(18) Synthetic primer 91 caggctgcgt ggggatgc 18 92 18 DNA Artificial Sequence misc_feature (1)..(18) Synthetic primer 92 ctggaggtgc tgcgcgtt 18 93 30 DNA Artificial Sequence misc_feature (1)..(30) Synthetic primer 93 cgcccccgcc cgctggctcc acgcagatgc 30 94 18 DNA Artificial Sequence misc_feature (1)..(18) Synthetic primer 94 cgtgaacagg gcgcatta 18 95 31 DNA Artificial Sequence misc_feature (1)..(31) Synthetic primer 95 cccccgcccg gcagcagaga tgttgttgga c 31 96 36 DNA Artificial Sequence misc_feature (1)..(36) Synthetic primer 96 ccgccgcccc cgccgccagg ctcctatctt gtgaca 36 97 21 DNA Artificial Sequence misc_feature (1)..(21) Synthetic primer 97 tgaagtcacc tgtgctgttg t 21 98 19 DNA Artificial Sequence misc_feature (1)..(19) Synthetic primer 98 ctacctgtgg gatctgggg 19 99 18 DNA Artificial Sequence misc_feature (1)..(18) Synthetic primer 99 tgctgaagct cacgctcc 18 100 20 DNA Artificial Sequence misc_feature (1)..(20) Synthetic primer 100 gggctcgtcg tcaatgcaag 20 101 40 DNA Artificial Sequence misc_feature (1)..(40) Synthetic primer 101 cgccgccccc gcccgccgcc caccacctgc agcccctcta 40 102 40 DNA Artificial Sequence misc_feature (1)..(40) Synthetic primer 102 gcggcccgcc gcccccgccg ccgcccagga cagcatcttc 40 103 18 DNA Artificial Sequence misc_feature (1)..(18) Synthetic primer 103 cgctgcccag catgttgg 18 104 24 DNA Artificial Sequence misc_feature (1)..(24) Synthetic primer 104 ggccggcagc ggcaaaggct tctc 24 105 19 DNA Artificial Sequence misc_feature (1)..(19) Synthetic primer 105 gcccagcacc agctcacat 19 106 21 DNA Artificial Sequence misc_feature (1)..(21) Synthetic primer 106 cgagccattt accacccata g 21 107 20 DNA Artificial Sequence misc_feature (1)..(20) Synthetic primer 107 ggcagccagc aggatctgaa 20 108 21 DNA Artificial Sequence misc_feature (1)..(21) Synthetic primer 108 ctgtgggcca gcagcaaggt g 21 109 21 DNA Artificial Sequence misc_feature (1)..(21) Synthetic primer 109 cctgaacctc cagcaccagc g 21 110 21 DNA Artificial Sequence misc_feature (1)..(21) Synthetic primer 110 aggtccaggg cgactcgctg g 21 111 21 DNA Artificial Sequence misc_feature (1)..(21) Synthetic primer 111 cagggccaca cgcgctgggc g 21 112 21 DNA Artificial Sequence misc_feature (1)..(21) Synthetic primer 112 ttggaggccc acgttgacct g 21 113 31 DNA Artificial Sequence misc_feature (1)..(31) Synthetic primer 113 cccccgcccg catgggtgtg gacgggtgag g 31 114 20 DNA Artificial Sequence misc_feature (1)..(20) Synthetic primer 114 taaaactgga tggggctctc 20 115 18 DNA Artificial Sequence misc_feature (1)..(18) Synthetic primer 115 ggcctccacc agcactaa 18 116 20 DNA Artificial Sequence misc_feature (1)..(20) Synthetic primer 116 gggtccccca gtccttccag 20 117 17 DNA Artificial Sequence misc_feature (1)..(17) Synthetic primer 117 tccccagccc gcccaca 17 118 20 DNA Artificial Sequence misc_feature (1)..(20) Synthetic primer 118 gccccctcac caccccttct 20 119 21 DNA Artificial Sequence misc_feature (1)..(21) Synthetic primer 119 tcccgctgct ccccccacgc a 21 120 18 DNA Artificial Sequence misc_feature (1)..(18) Synthetic primer 120 gatgccgtgg ggaccgtc 18 121 20 DNA Artificial Sequence misc_feature (1)..(20) Synthetic primer 121 gtgagcaggt ggcagtctcg 20 122 21 DNA Artificial Sequence misc_feature (1)..(21) Synthetic primer 122 ccaccccctc tgctcgtagg t 21 123 19 DNA Artificial Sequence misc_feature (1)..(19) Synthetic primer 123 ggtcccaagc acgcatgca 19 124 22 DNA Artificial Sequence misc_feature (1)..(22) Synthetic primer 124 tgccggcctc ctgcgctgct ga 22 125 29 DNA Artificial Sequence misc_feature (1)..(29) Synthetic primer 125 gcgggcaggg tgagcaggtg gggccatcc 29 126 26 DNA Artificial Sequence misc_feature (1)..(26) Synthetic primer 126 gaggctgtgg gggtccagtc aagtgg 26 127 25 DNA Artificial Sequence misc_feature (1)..(25) Synthetic primer 127 agggaggcag aggaaagggc cgaac 25 128 29 DNA Artificial Sequence misc_feature (1)..(29) Synthetic primer 128 cgtcccgcct gcactgacct cacgcatgt 29 129 41 DNA Artificial Sequence misc_feature (1)..(41) Synthetic primer 129 cggcccgccg cccccgcccg gccaaaggga aagggattgg a 41 130 21 DNA Artificial Sequence misc_feature (1)..(21) Synthetic primer 130 ccgcggagcc tgctgtgcta t 21 131 39 DNA Artificial Sequence misc_feature (1)..(39) Synthetic primer 131 ccgccgcccc cgcccgcttg gtggagacgg tgtagttgc 39 132 21 DNA Artificial Sequence misc_feature (1)..(21) Synthetic primer 132 tccaatccct ttccctttgg c 21 133 22 DNA Artificial Sequence misc_feature (1)..(22) Synthetic primer 133 cagcagccca tgaaacagaa ag 22 134 21 DNA Artificial Sequence misc_feature (1)..(21) Synthetic primer 134 tatgctttca ggcccgtggc a 21 135 23 DNA Artificial Sequence misc_feature (1)..(23) Synthetic primer 135 agagcccata cccggtccag tcc 23 136 23 DNA Artificial Sequence misc_feature (1)..(23) Synthetic primer 136 ggactggacc gggtatgggc tct 23 137 31 DNA Artificial Sequence misc_feature (1)..(31) Synthetic primer 137 cccccgcccg cacccaggcc ctcctcgact c 31 138 30 DNA Artificial Sequence misc_feature (1)..(30) Synthetic primer 138 cccccgccgc tgggtgggct cggctctatc 30 139 23 DNA Artificial Sequence misc_feature (1)..(23) Synthetic primer 139 tggtagcgat gctcacgtca ctt 23 140 23 DNA Artificial Sequence misc_feature (1)..(23) Synthetic primer 140 caggccaaag ctgagatgac ttg 23 141 20 DNA Artificial Sequence misc_feature (1)..(20) Synthetic primer 141 agaggcgcag gagggaggtc 20 142 18 DNA Artificial Sequence misc_feature (1)..(18) Synthetic primer 142 ccctctgccc ccgcattg 18 143 21 DNA Artificial Sequence misc_feature (1)..(21) Synthetic primer 143 aagcgcaaaa gggctgcgtc g 21 144 22 DNA Artificial Sequence misc_feature (1)..(22) Synthetic primer 144 ggccctccct gccttctagg cg 22 145 20 DNA Artificial Sequence misc_feature (1)..(20) Synthetic primer 145 ccgtgctgtg tggaggagag 20 146 21 DNA Artificial Sequence misc_feature (1)..(21) Synthetic primer 146 cctcttcctg cccagccctt c 21 147 21 DNA Artificial Sequence misc_feature (1)..(21) Synthetic primer 147 cttcccgagc agcctttggt g 21 148 20 DNA Artificial Sequence misc_feature (1)..(20) Synthetic primer 148 ctgagctgcc gcccgctgac 20 149 20 DNA Artificial Sequence misc_feature (1)..(20) Synthetic primer 149 aggaccccca gcccagccca 20 150 18 DNA Artificial Sequence misc_feature (1)..(18) Synthetic primer 150 cttggcgcag cttggact 18 151 20 DNA Artificial Sequence misc_feature (1)..(20) Synthetic primer 151 acacccagca aggacacgca 20 152 20 DNA Artificial Sequence misc_feature (1)..(20) Synthetic primer 152 tgtgacacat cccctggtac 20 153 20 DNA Artificial Sequence misc_feature (1)..(20) Synthetic primer 153 gcaagggtga gcttcagagc 20 154 51 DNA Artificial Sequence misc_feature (1)..(51) Synthetic primer 154 gccccgcgcc cgtcccgccg cccccgcccg accctatgcc tcctgtacct c 51 155 18 DNA Artificial Sequence misc_feature (1)..(18) Synthetic primer 155 cccctcctct ggcaatcc 18 156 20 DNA Artificial Sequence misc_feature (1)..(20) Synthetic primer 156 cctgccggga gcacgacgag 20 157 20 DNA Artificial Sequence misc_feature (1)..(20) Synthetic primer 157 ctgggctggg gcacggcggg 20 158 21 DNA Artificial Sequence misc_feature (1)..(21) Synthetic primer 158 gggggctacc acggcgcggg c 21 159 20 DNA Artificial Sequence misc_feature (1)..(21) Synthetic primer 159 ttggggcgtt catttggatc 20 160 20 DNA Artificial Sequence misc_feature (1)..(20) Synthetic primer 160 accacacaga aataggaggg 20 161 24 DNA Artificial Sequence misc_feature (1)..(24) Synthetic primer 161 ttgttattgt tttaattgtt ctta 24 162 25 DNA Artificial Sequence misc_feature (1)..(25) Synthetic primer 162 ctactctgac taaatttttc ttctt 25 163 20 DNA Artificial Sequence misc_feature (1)..(20) Synthetic primer 163 tttggttttg tattgtggtg 20 164 23 DNA Artificial Sequence misc_feature (1)..(23) Synthetic primer 164 aaggatttac gaagtttaaa ttg 23 165 21 DNA Artificial Sequence misc_feature (1)..(21) Synthetic primer 165 agaacctcag gaagcatgat t 21 166 20 DNA Artificial Sequence misc_feature (1)..(20) Synthetic primer 166 taggtaccaa atcaaatccg 20 167 20 DNA Artificial Sequence misc_feature (1)..(20) Synthetic primer 167 gtctcagtgt tctgctcctc 20 168 22 DNA Artificial Sequence Sequence misc_feature (1)..(22) Synthetic primer 168 aaatacaact gtcagcaaca ta 22 

1. A method of mutation analysis of a target nucleic acid, said method comprising: incubating a sample comprising said target nucleic acid in a reaction mixture, in the presence of at least one first nucleic acid and at least one second nucleic acid, wherein said first nucleic acid comprises a primer sequence which anneals to a unique site of a sequence of SEQ ID NO. 1 or 2, and said second nucleic acid has an opposite orientation from said first nucleic acid, and wherein said incubation produces amplified products; generating duplexes in said amplified products; and detecting the presence or absence of a heteroduplex from said duplexes, wherein the presence of a heteroduplex indicates the presence of a potential mutation in said target nucleic acid, and wherein the absence of a heteroduplex indicates the absence of a mutation in said target nucleic acid.
 2. The method of claim 1, the method further comprising determining the sequence of a heteroduplex region; and comparing the sequence of the heteroduplex region to SEQ ID NO. 1 or 2; wherein a sequence difference in the heteroduplex region compared to SEQ ID NO. 1 or 2 resulting in a predicted functional change in the protein encoded by said target nucleic acid is indicative of a mutation in said target nucleic acid.
 3. The method of claim 1, wherein said first or second nucleic acid comprises a sequence selected from the group consisting of SEQ ID NOs. 3-49.
 4. The method of claim 1, said method further comprising performing a nested amplification reaction using said amplified products generated by said first and second nucleic acids as templates and generating duplexes in amplified products from said nested amplification.
 5. The method of claim 4, wherein said nested amplification reaction is performed using at least one primer selected from the group consisting of SEQ ID NOs. 3-49 and their complementary sequences.
 6. The method of claim 1, wherein identifying the presence or absence of a heteroduplex from said duplexes is performed by DHPLC.
 7. The method of claim 1, wherein the sequence of the heteroduplex region is determined by DNA sequencing.
 8. The method of claim 1, wherein said second nucleic acid comprises a primer sequence which anneals to a unique site within a sequence of SEQ ID NO. 1 or
 2. 9. The method of claim 1, wherein said sample comprising said target template is selected from the group consisting of: genomic DNA, cDNA, total RNA, mRNA, and a cell sample.
 10. The method of claim 1, wherein said incubating comprises an amplification reaction selected from the group consisting of: a polymerase chain reaction, a ligase chain reaction (LCR) and a nucleic acid-specific based amplification.
 11. The method of claim 1, further comprising confirming the amplified product is a PKD-specific product with one or more restriction enzymes.
 12. The method of claim 11, wherein said restriction enzyme cleaves a PKD-specific product to generate a digestion pattern distinguishable from a PKD homologue product.
 13. The method of claim 11, wherein said restriction enzyme is selected from the group consisting of: Pst I, Stu I, Xma I, Mlu I, Pvu II, BssHII, Fsp I, Msc I, and Bln I.
 14. A diagnosis method for identifying a patient affected with PKD, said method comprising: (c) obtaining a sample from an individual; (d) incubating said sample in a reaction mixture, in the presence of at least one first nucleic acid and at least one second nucleic acid, wherein said first nucleic acid comprises a primer sequence which anneals to a unique site within a sequence of SEQ ID NO. 1 or 2, and said second nucleic acid has an opposite orientation from said first nucleic acid, and wherein said incubation produces amplified products; (c) generating duplexes in said amplified products; (d) detecting the presence or absence of a heteroduplex from said duplexes, and(e) determining the sequence of the heteroduplex region wherein the presence of a mutation in the heteroduplex region as compared to SEQ ID No. 1 or 2 is indicative that said individual is affected with PKD.
 15. The method of claim 14, wherein said detection of a hereroduplex is performed by DHPLC.
 16. The method of claim 14, wherein said sequence is determined by DNA sequencing.
 17. The method of claim 14, wherein said second nucleic acid comprises a primer sequence which anneals to a unique site within a sequence of SEQ ID NO. 1 or
 2. 18. The method of claim 14, wherein said first or second nucleic acid comprises a primer sequence selected from the group consisting of SEQ ID NOs. 3-49.
 19. The method of claim 14, said method further comprising performing a nested amplification reaction using said amplified products generated by said first and second nucleic acids as templates and generating duplexes from said nested amplification.
 20. The method of claim 19, wherein said nested amplification reaction is performed using at least one primer selected from the group consisting of SEQ ID NOs. 3-49 and their complementary sequences.
 21. The method of claim 14, wherein said sample is selected from the group consisting of: a genomic DNA, cDNA, total RNA, mRNA, and a cell.
 22. The method of claim 14, wherein said amplification reaction is selected from the group consisting of: a polymerase chain reaction, a ligase chain reaction (LCR) and a nucleic acid-specific based amplification.
 23. The method of claim 14, further comprising verifying a said specifically amplified product with one or more restriction enzymes.
 24. The method of claim 23, wherein said restriction enzyme cleaves a PKD-specific product to generate a digestion pattern distinguishable from a PKD homologue product.
 25. The method of claim 24 wherein said restriction enzyme is selected from the group consisting of: Pst I, Stu I, Xma I, Mlu I, Pvu II, BssHII, Fsp I, Msc I, and Bln I. 